Caspase inhibitors containing isoxazoline ring

ABSTRACT

The present invention relates to an isoxazoline derivative as an inhibitor against various caspases, a process for preparing the same, and a therapeutic composition for preventing inflammation and apoptosis comprising the same

TECHNICAL FIELD

The present invention relates to an isoxazoline derivative as aninhibitor against various caspases including caspase-1[interleukin-1βconverting enzyme, ICE] and caspase-3[apopain/CPP-32], a process forpreparing the same, and a therapeutic composition for preventinginflammation and apoptosis comprising the same.

BACKGROUND ART

Caspase is a new kind of cysteine protease in the form of α₂β₂discovered during the last 10 years. About 14 kinds thereof have beenknown until now. Caspase-1 (ICE), one of them, is a kind of cytokinesand participates in converting the inactive prointerleukin-1β to theactive interleukin-1β. Interleukin-1 consists of interleukin-1α andinterleukin-1β, both of which are synthesized in monocytes in the formof a precursor having 31 kDa. Only prointerleukin-1β is activated byICE. The positions hydrolyzed by caspase-1 are Asp²⁷-Gly²⁸ andAsp¹¹⁶-Ala¹¹⁷. The hydrolysis of the latter position givesinterleukin-1β. Interleukin-1β has been reported to act as an importantmediator in causing inflammation (1,3). Caspase-1 has been discoveredfor the first time in 1989, and in two independent study groups, thethree dimensional structure thereof was determined by X-raycrystallographic method.

Caspase-3(CPP-32) is broadly studied for its role or mechanism foraction, and its three dimensional structure was determined in 1996(2).Caspase-3(apopain) activated from procaspase-3 hydrolyzes(P4)Asp-X-X-Asp(P1) motif, and the known substrates includepoly(ADP-ribose) polymerase, U1 70,000 Mr small nuclearribonucleoprotein and catalytic subunit of 460,000 Mr DNA-dependentprotein kinase, etc. The X-ray structure of caspase-7 has been reportedto be very similar to that of caspase-3(4).

Caspase-8 and 9 are present in the upstream of caspase-3,6,7, and thesecaspases are known to participate in the apoptosis cascade. The X-raystructure of caspase-8 was determined in 1999(5), and particularly theinhibitors thereof may be advantageously used for treating the diseasesrelated to apoptosis.

Caspase inhibitors mean those compounds that inhibit the activity ofcaspase, and so control such symptoms as inflammation, apoptosis, etc.caused by the caspase activity. Diseases or symptoms that may be treatedor attenuated by administering the inhibitors include the following:reumatoid arthritis, inflammatory bowel disease, graft vs. host disease,sepsis, osteoarthritis, osteoporosis, acute and chronic myelogenousleukemia, meningitis, salpingitis, septic shock, cholangitis, colitis,encephalitis, endocarditis, glomerulonephritis, hepatitis, myocarditis,pancreatitis, type I diabetes mellitus, multiple sclerosis, Alzheimer'sdisease, Parkinson's disease, hepatocirrhosis(6).

Among the caspase inhibitors known until now, the most notedirreversible inhibitors are the following:

Both the above inhibitors exhibit their activity based on the commonmechanism that they irreversibly inactivate the enzyme to suppress thecell apoptosis (irreversible, broad-spectrum inhibitor). It has beenreported that irreversible inhibitor has much more effective inhibitoryactivity when comparing the irreversible and reversible inhibitors (7).Both IDN-1965 of IDUN Co. and MX-1013 of Maxim Co. are reported to showactivity in cell apoptosis model for hepatic injury(8, 9). Thesecompounds are now in the stage of preclinical test. The irreversibleinhibitor IDN-6556, the structure of which has been recently reported,is now in the stage of phase II clinical test as a therapeutic agent forhepatic injury(10, 11).

REFERENCES

(1) Inflammation: Basic Principles and Clinical Correlates, 2nd ed., edby Gallin, Goldstein and Snyderinan. Raven Press Ltd., New York. 1992,pp 211-232; Blood, 1996, 87(6), 2095-2147.

(2) Wilson, K. P. et al, Nature, 1994,370. 270; Walker, N. P. C. et al.Cell, 1994, 78, 343; Nature Structural Biology, 1996, 3(7), 619.

(3) Thornberry, N. A. et al, Nature, 1992, 356.768; NatureBiotechnology, 1996, 14, 297; Protein Science, 1995, 4, 3; Nature, 1995,376(July 6), 37; Protein Science, 1995, 4, 2149.

(4) Wei, Y. et al, Chemistry and Biology, 2000, 7, 423.

(5) Blanchard H. et al, Structure, 1999, 7, 1125; Blanchard H. et al, J.of Mol. Biol., 2000, 302, 9.

(6) References for caspase related diseases

Dementia: Arch Neurol March 2003; 60(3):369-76, Caspase gene expressionin the brain as a function of the clinical progression of Alheimerdisease. Pompl P N, Yemul S, Xiang Z, Ho L, Haroutunian V, Purohit D,Mohs R, Pasinetti G M.

Cerebral stroke: Proc Natl Acad Sci USA Nov. 12, 2002; 99(23):15188-93,Caspase activation and neuroprotection in caspase-3-deficient mice afterin vivo cerebral ischemia and in vitro oxygen glucose deprivation. Le DA, Wu Y, Huang Z, Matsushita K, Plesnila N, Augustinack J C, Hyman B T,Yuan J, Kuida K, Flavell R A, Moskowitz M A.

Brain impairment due to AIDS: J Neurosci May 15, 2002; 22(10):4015-24,Caspase cascades in human immunodeficiency virus-associatedneurodegeneration. Garden G A, Budd S L, Tsai E, Hanson L, Kaul M,D'Emilia D M, Friedlander R M, Yuan J, Masliah E, Lipton S A.

Diabetes: Diabetes June 2002; 51(6):1938-48, Hyperglycemia-inducedapoptosis in mouse myocardium: mitochondrial cytochrome C-mediatedcaspase-3 activation pathway. Cai L, Li W, Wang G, Guo L, Jiang Y, KangY J.

Gastric ulcer: J Physiol Pharmacol December 1998; 49(4):489-500, Role ofbasic fibroblast growth factor in the suppression of apoptotic caspase-3during chronic gastric ulcer healing. Slomiany B L, Piotrowski J,Slomiany A.

Cerebral injure by hepatitis: J Viral Hepat March 2003; 10(2):81-6,Cerebral dysfunction in chronic hepatitis C infection. Forton D M,Taylor-Robinson S D, Thomas H C.

Fulminant hepatic failure: Gastroenterology August 2000; 119(2):446-60,Tumor necrosis factor alpha in the pathogenesis of human and murinefulminant hepatic failure. Streetz K, Leifeld L, Grundmann D, RamakersJ, Eckert K, Spengler U, Brenner D, Manns M, Trautwein C.

Sepsis: Nat Immunol December 2000; 1(6):496-501, Caspase inhibitorsimprove survival in sepsis: a critical role of the lymphocyte. HotchkissR S, Chang K C, Swanson P E, Tinsley K W, Hui J J, Klender P,Xanthoudakis S, Roy S, Black C, Grimm E, Aspiotis R, Han Y, Ncholson DW, Karl I E.

Organ transplantation rejection: Xenotransplantation May 2001;8(2):115-24, In vitro prevention of cell-mediated xeno-graft rejectionvia the Fas/FasL-pathway in CrmA-transducted porcine kidney cels. FujinoM, Li X K, Suda T, Hashimoto M, Okabe K, Yagnuma H, Mikoshiba K, Guo L,Okuyama T, Enosawa S, Amemiya H, Amano T, Suzuki S.

Rheumatic arthritis: Prog Med Chem 2002;39:1-72, Caspase inhibitors asanti-inflammatory and antiapoptotic agents. Graczyk P P.

Ischemic cardiac diseases: Am J Physiol Heart Circ Physiol September2002; 283(3):H990-5, Hypoxia-induced cleavage of caspase-3 andDFF45/ICAD in human failed cardiomyocytes. Todor A, Sharov V G, TanhehcoE J, Silverman N, Bernabei A, Sabbah H N.

Anti-inflammation: J Immunol Mar. 15, 2003; 170(6):3386-91, Abroad-spectrum caspase inhibitor attenuates allergic airway inflammationin murine asthma model. Iwata A, Nshio K, Winn R K, Chi E Y, Henderson WR Jr, Harlan J M.

Cirrhosis: i) J Pharmaccl Exp Ther. March 2004; 308(3):1191-6, Thecaspase inhibitor Idn-6556 attenuates hepatic injury and fibrosis in thebile duct ligated mouse. Canbay A., Fledstein A., Baskin-Bey E., Bronk FS. Gores G J.; ii) Hepatology. February 2004;39(2):273-8, Apoptosis: thenexus of liver injury and fibrosis. Canbay A, Friedman S, Gores G J.;iii) Hepatology. November 2003; 38(5):1188-98, Kupffer cell engulfmentof apoptotic bodies stimulates death ligand and cytokine expression.Canbay A, Feldstein A E, Higuchi H, Werneburg N, Grambihler A, Bronk SF, Gores G J.

(7) Wu J. et al, Methods: A Companion to Methods in Enzymology, 1999,17, 320.

(8) Hoglen N. C. et al, J. of Pharmacoloy and Experimental Therapeutics,2001, 297, 811.

(9) Jaeschke H. et al, Toxicology and Applied Pharmacology, 2000, 169,77.

(10) Hoglen N. C. et al, J. Phannacol Exp. Ther., 2004, 309(2):634.Characterization of IDN-6556(3-[2-(2-tert-butyphenylaminooxalyl)-amino]-propionylamino]-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoicacid): a liver-targeted caspase inhibitor;

(11) Canbay A et al, J. Pharmacol. Exp. Ther., 2004, 308(3), 1191. Thecaspase inhibitor IDN-6556 attenuates hepatic injury and fibrosis in thebile duct ligated mouse.

DISCLOSURE OF THE INVENTION

The present inventors newly designed and synthesized some compoundswhich can be used as an effective inhibitor against caspases and have adistinctive structure and high selectivity for similar enzymes, anddetermined their binding ability and inhibitory activity for caspases.As a result, the inventors have discovered that a compound of thefollowing formula (1) does meet such requirements, and completed thepresent invention.

in which

A, B, R, R¹, R² and X are defined as described below.

Therefore, the present invention provides the novel isoxazocinederivative of formula (1), salt, or stereoisomer thereof havingeffective inhibitory activity against caspases.

It is another object of the present invention to provide a process forpreparing the compound of formula (1), salt, or stereoisomer thereof.

It is also another object of the present invention to provide acomposition, a use, or a method for inhibiting caspases, specifically atherapeutic composition, a use, or a method for preventing inflammationand apoptosis, comprising the compound of formula (1), salt, orstereoisomer thereof as an active ingredient together with thepharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibitory activity of Compound (Iii) against thehepatic fibrosis due to the bile stagnation: “a)” in FIG. 1 representsthe case of Sham operation, “b)” represents the case of BDL operationwhere only the vehicle was administered after the ligation of biliaryduct, “c)” represents the case where Compound (Iii) was orallyadministered in a dosage of 3 mg/kg twice a day for 1 week after 1 weekfrom the ligation of biliary duct, and “d)” represents the case whereCompound (Iii) was orally administered in a dosage of 10 mg/kg twice aday for 1 week after 1 week from the ligation of biliary duct.

FIG. 2 shows the inhibitory activity of Compound (Iii) against thehepatic cell apoptosis due to the bile stagnation: “a)” in FIG. 2represents the case of Sham operation, “b)” represents the case of BDLoperation where only the vehicle was administered after the ligation ofbiliary duct, “c)” represents the case where Compound (Iii) was orallyadministered in a dosage of 3 mg/kg twice a day for 1 week after 1 weekfrom the ligation of biliary duct, “d)” represents the case whereCompound (Iii) was orally administered in a dosage of 10 mg/kg twice aday for 1 week after 1 week from the ligation of biliary duct, and “e)”is a graph showing the number of hepatic cels subjected to apoptosis ineach treatment group.

BEST MODE FOR CARRYING OUT THE INVENTION

In advance of illustrating the present invention in detail, thefollowing important terms are defined first:

a) Simple Alkyl Chain (SAC, below) means a hydrocarbon having 1 to 8carbon atoms in either linear or branched isomeric form.

b) Simple Cyclo Alkyl Chain (SCAC, below) means a cyclic radical having3 to 10 carbon atoms.

c) Aryl group (Ar, below) includes both the aromatic and heteroaromaticgroups. The aromatic group means a 5 to 15-membered single or fusedunsaturated cycle. One or more hydrogens may be replaced with a group(s)selected from the following: acyl, amino, carboalkoxy, carboxy,carboxyamino, cyano, halo, hydroxy, nitro, thiol, alkyl, cycloalkyl,alkoxy, aryloxy, sulfoxy, and guanido group. The heteroaromatic groupmeans the aromatic group containing 1 to 5 hetero atoms selected from agroup consisting of oxygen, sulfur, and nitrogen. Likewise, one or morehydrogens may be replaced with a group(s) selected from the following:acyl, amino, carboalkoxy, carboxy, carboxyamino, cyano, halo, hydroxy,nitro, thiol, alkyl, cycloalkyl, alkoxy, aryl, aryloxy, sulfoxy, andguanido group. Or, for example, in the case of pyridyl, an alkyl groupcan be added to the nitrogen atom to convert the pyridyl group to apyridinium group having (+)-charge on the nitrogen atom.

The aryl group includes phenyl, biphenyl, 1-naphthyl, 2-naphthyl,pyridinyl, N-alky-pyridinium, pyrimidinyl, quinolinyl, benzothienyl,indolyl, pyrazinyl, isoindolyl, isoquinolyl, quinazolinyl, quinoxalinyl,phthalazinyl, imidazolinyl, isoxazolyl, pyrazolyl, oxazolyl, thiazolyl,indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl,thienyl, pyrrolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl,oxazolopyridinyl, imidazopyridinyl, isothiazolyl, cinnolinyl,carbazolyl, isochromanyl, chromanyl, tetrahydroisoquinolinyl,isoindolinyl, isobenzotetrahydrofuranyl, isobenzotetrahydro-thienyl,isobenzothienyl, benzoxazolyl, pyridopyridinyl, benzotetrahy-drofuranyl,benzotetrahydrothienyl, purinyl, benzodioxolyl, triazinyl, phenoxazinyl,phenothiazinyl, pteridinyl, benzothiazolyl, imidazopyridinyl,imidazothiazolyl, dihydrobenzisoxazinyl, benzisoxazinyl, benzoxazinyl,dihydrobenzisothiopyranyl, benzopyranyl, benzothiopyranyl, coumarinyl,isocoumarinyl, chromonyl, chromanonyl, pyridinyl-N-oxide,tetrahydroquinolinyl-N-oxide, dihydroquinolinyl, dihydroquinolinonyl,dihydroisoquinolinonyl, dihydrocoumarinyl, dihydroisocoumarinyl,isoindolinyl, benzodioxanyl), benzoxolinonyl, N-alky-pyridium,pyrrolyl-N-oxide, pyrimidinyl-N-oxide, pyrazinyl-N-oxide,quinolinyl-N-oxide, indolyl-N-oxide, indolinyl-N-oxide,isoquinolyl-oxide, qunazolinyl-N-oxide, quinoxalinyl-N-oxide,phthalazinyl-N-oxide, imidazolinyl-N-oxide, isoxazolyl-N-oxide,oxazolyl-N-oxide, thiazolyl-N-oxide, indolizinyl-N-oxide,indazolyl-N-oxide, benzothiazolyl-N-oxide, benzimidazolyl-N-oxide,pyrrolyl-N-oxide, oxadiazolyl-N-oxide, thiadiazolyl-N-oxide,triazolyl-N-oxide, tetrazolyl-N-oxide, etc.

d) Simple Alkyl Chain substituted by Aryl (SAC—Ar, below) means astraight-chain or branched alkyl which has 1 to 8 carbon atoms and issubstituted by the above mentioned aryl group.

e) Natural amino acid includes the following: Glycine, Alanine, Valine,Leucine, Isoleucine, Serine, Threonine, Cysteine, Methionine, Proine,Aspartic acid, Asparagine, Glutamic acid, Glutamine, Lysine, Arginine,Histidine, Phenylalanine, Tyrosine, and Tryptophan

f) The protecting group of simple ester is a hydrocarbon having 1 to 8carbon atoms in either linear or branched isomeric form.

Further, the present specification includes the following abbreviations:

N-chlorosuccinimide: NCS

N-methylmorpholine: NMM

O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate:HATU

N,N-dimethyl formamide: DMF

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide: EDC

1-hydroxybenzotriazole hydrate: HOBt

trifluoroacetic acid: TFA

t-butoxycarbonyl: Boc

benzyloxycarbonyl: Cbz

methyl: Me

ethyl: Et

equivalent: Eq

The substituents included in the above formula (1) are specificallydefined as follows.

I) R represents H, simple alkyl chain (—SAC), simple cycloalkyl chain(—SCAC), aryl group (—Ar), or simple alkyl chain substituted by aryl(—SAC—Ar),

II) R¹ represents —SAC, —SCAC, —Ar, —SAC—Ar, or a side chain residue ofall the natural amino acids; and the compound of formula (1) may existin a specific diastereomeric form, or mixtures thereof when the carbonto which R¹ is attached becomes a stereocenter due to the R¹ group; orthe compound of formula (1) may have a protecting group in an ester form(—CO₂R³ wherein R³ is —SAC) or a sulfonamide form (—CONHSO₂R⁴ wherein R⁴is —SAC), or may exist in the form of pharmaceutically acceptable salt,when R¹ is a side chain residue of an amino acid containing carboxylmoiety; or the compound of formula (1) may also exist in the form ofpharmaceutically acceptable salt when R¹ is a side chain residue of anamino acid containing a base moiety,

III) R² represents —SAC, —SCAC, —Ar, —SAC—Ar, or a side chain residue ofthe natural amino acids; and the compound of formula (1) may exist in aspecific diastereomeric form, or mixtures thereof when the carbon towhich R² is attached becomes a stereocenter due to the R² group; thecompound of formula (1) may have a protecting group in an ester form(—CO₂R⁵ wherein R⁵ is —SAC) or a sulfonamide form (—CONSO₂R⁶ wherein R⁶is —SAC), or may exist in the form of pharmaceutically acceptable salt,when R² is a side chain residue of an amino acid containing carboxylmoiety; or the compound of formula (1) may also exist in the form ofpharmaceutically acceptable salt when R² is a side chain residue of anamino acid containing a base moiety, or R² further represents H; —(CH)OR⁷ wherein R⁷ is —SAC, —SCAC, —Ar, or —SAC—Ar, and n=1 or 2; or—(CH₂)_(n)OC(═O)R⁸ wherein R⁸ is —SAC, —SCAC, —Ar, or —SAC—Ar, and n=1or 2,

IV) A represents —(CH₂)_(n)-(n=0-4), —O—(CH₂)_(n)-(n=0-4), or—NR⁹—(CH₂)_(n)-(n=0-4) wherein R⁹ is —SAC, —SCAC, —Ar, or —SAC—Ar,

V) Brepresents H, —SAC, —SCAC, —Ar, or —SAC—Ar, or

VI) R and R¹ may form a cycle together with the carbon atom to whichthey are attached, where —R—R¹— is —(CH₂)_(n), —(CH₂)_(n)—O—(CH₂)_(m)—,or —(CH₂)_(n)—NR¹⁰—(CH₂)_(m)— wherein n+m<9 and R¹⁰ is —SAC, —SCAC, —Ar,—SAC—Ar, —C(═O)—SAC, —C(═O)—SCAC, —C(═O)—Ar, or —C(═O)—SAC—Ar,

VII) X represents —C(═O)CH₂OR¹¹ wherein R¹¹ is —SAC, —SCAC, —Ar, or—SAC—Ar; —C(═O)CH₂OC(═O)R¹² wherein R¹² is —SAC, —SCAC, —Ar, or —SAC—Ar;—CH═CH—CO₂R¹³ wherein R¹³ is —SAC, —SCAC, —Ar, or —SAC—Ar; —CH═CH—SO₂R¹⁴wherein R¹⁴ is —SAC, —SCAC, —Ar, or —SAC—Ar; —C(═O)CH═CH₂; or —COCH₂—Wwherein W is —N₂, —F, —Cl, —Br, —I, —NR¹⁵R¹⁶(R¹⁵ and R¹⁶ each are —SAC,—SCAC, —Ar, or —SAC—Ar, or together may form 3- to 6-membered saturatedor unsaturated cyclic group), —SR¹⁷ (R¹⁷ is —SAC, —SCAC, —Ar, or—SAC—Ar), or is the following formula:

wherein

Y is H, —OH, —OR¹⁸ (R¹⁸═—SAC or —SCAC), —C(═O)R¹⁹(R¹⁹═—H, —SAC, or—SCAC), —F, —Cl, —Br, —I, —CN, —NC, —N₃, —CO₂H, CF₃, —CO₂R²⁰(R²⁰═—SAC or—SCAC), —C(═O)NHR²¹(R²¹═—SAC or —SCAC), or C(═O)R²²R²³(R²² and R²³ eachare —SAC, —SCAC, —Ar, or —SAC—Ar),

R²⁴ is H, —SAC, —SAC—Ar, or —Ar.

The preferred compounds among the compound of formula (1) above arethose wherein

R represents H;

R¹ represents —CH₂COOH, —CH₂COOR³(R³═SAC), or —CH₂CONHSO₂R⁴(R⁴═SAC;

R² represents H, —SAC, —Ar, or —(CH₂)_(n)OR⁷(R⁷═—SAC, —SCAC, —Ar, or—SAC—Ar, and n=1 or 2); or

X represents —C(═O)CH₂O Ar, —C(═O)CH₂OC(═O)Ar, or —COCH₂—W wherein W is—N₂, —F, —Cl, —Br, —I, —NR¹⁵R¹⁶(R¹⁵ and R¹⁶ each are —SAC, —SCAC, —Ar,or —SAC—Ar, or together may form 3- to 6-membered saturated orunsaturated cyclic group), or —SR¹⁷(R¹⁷ is —SAC, —SCAC, —Ar, or—SAC—Ar).

More preferred compounds among the compound of formula (1) above arethose wherein

R represents H,

R¹ represents —CH₂COOH, —CH₂COOR³(R═SAC), or —CH₂CONHSO₂R⁴(R⁴═SAC),

III) R² represents H, —SAC, —Ar, or —(CH₂)_(n)OR⁷(R⁷═—SAC, —SCAC, —Ar,or —SAC—Ar, and n=1 or 2),

IV) A represents —(CH₂)_(n)-(n=0-4) or —O—(CH₂)_(n)(n=0-4),

V) B represents H, —SAC, —SCAC, —Ar, or —SAC—Ar,

VI) X represents —COCH₂N₂, —COCH₂F, —COCH₂Cl, —COCH₂Br, —COCH₂I,—COCH₂OAr, —COCH₂OCOAr or —COCH₂SR¹⁷(R¹⁷ is —SAC, —SCAC, —Ar or—SAC—Ar).

Particularly preferred compounds are those selected from the followinggroup:

(1)(3S)-5-[(2,6-dichlorobenzoyl)oxy]-3-({[5-methyl-3-phenyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa);

(2)(3S)-3-({[5-methyl-3-phenyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-phenoxypentanoicacid (Ibb);

(3)(3S)-3-({[5-ethyl-3-phenyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Icc);

(4)(3S)-3-({[5-ethyl-3-(1-naphthyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Idd);

(5)(3S)-3-({[5-ethyl-3-(2-naphthyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Iee);

(6)(3S)-3-({[5-ethyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Iff);

(7)3-({[5-ethyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Igg);

(8) ethyl3-({[5-ethyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoate(Ihh);

(9) 5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iii);

(10)3-({[5-ethyl-3-(4-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Ijj);

(11)3-({(3-(benzothiophen-2-yl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Ikk);

(12) (3S)-3-({[3-(1,3-dimethyl-1H-indol-2-yl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid ( Ill);

(13) 3-({[3-(1,3-dimethyl-1H-indol-2-yl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Imm);

(14)(3S)-3-({[5-ethyl-3-(1-naphthylmethyl)-4,5-dihydro-5-isoxazolyl]carbonyl{amino)-4-oxo-5-(2,3,5,6-tetrafluoropheoxy)pentanoicacid ( Inn);

(15)(3S)-5-[(2,6-dichlorobenzoyl)oxy]-3-[({5-ethyl-3-[2-(1naphthyl)ethyl]-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-4-oxopentanoicacid (Ioo);

(16)(3S)-3-[({5-ethyl-3-[(1-naphthyloxy)methyl]-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Ipp);

(17)(3S)-3-{[(3-{[(4-chloro-1-naphthyl)oxy]methyl}-5-ethyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid (Iqq);

(18)(3S,4E)-6-ethoxy-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-6-oxo-4-hexenoicacid (Irr);

(19)(3S,4E)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-(methylsulfonyl)-4-pentenoicacid (Iss);

(20) 5-fluoro-3-({[(5S)-3-(1-isoquinolinyl)-5-propyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Itt);

(21)3-({[(5S)-5-ethyl-3-(1-naphthyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iuu);

(22)3-({[(5S)-5-ethyl-3-(2-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Ivv);

(23)3-({[(5R)-5-ethyl-3-(3-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iww);

(24)3-({[5-ethyl-3-(8-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Ixx);

(25)3-({[5-ethyl-3-(3-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iyy);

(26) 5-fluoro-3-({[(5R)-5-isopropyl3-(2-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Izz);

(27)3-({[5-ethyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa1);

(28)3-[({3-[3-(tert-butyl)phenyl]-5-ethyl4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa2);

(29)3-[({3-[4-(tert-butyl)phenyl]-5-ethyl-}4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa3);

(30) 5-fluoro-3-({[(5R)-5-isopropyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa4);

(31)3-[({(5R)-3-[3-(tert-butyl)phenyl]-5-isopropyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa5);

(32)3-{[(3-[1,1′-biphenyl]-3-yl-5-isopropyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-5-fluoro-4-oxopentanoicacid (Iaa6);

(33)3-({[5-ethyl-3-(2-pyridinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa7);

(34)3-[({3-[4-(tert-butyl)-2-pyridinyl]-5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa8);

(35)3-[({(5R)-3-[4-(tert-butyl)-2-pyridinyl]-5-isopropyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa9);

(36)3-({[5-ethyl-3-(4-isobutyl-2-pyridinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa10); (37)3-({[3-(4-acetyl-2-pyridinyl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa11);

(38)3-({[3-(4-cyclopropyl-2-pyridinyl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa12);

(39)3-({[3-(4-cyclopentyl-2-pyridinyl)-5-ethyl4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa13);

(40)3-({[(5R)-3-(4-cyclopentyl-2-pyridinyl)-5-isopropyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa14);

(41)3-({[3-(4-cyclohexyl-2-pyridinyl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa15)

(42)3-({[5-ethyl-3-(5,6,7,8-tetrahydro-1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa16);

(43)5-fluoro-3-({[5-isopropyl-3-(4-phenyl-2-pyridinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa17);

(44)(3S)-5-[(diphenylphosphoryl)oxy]-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa 18);

(45)(3S)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-({[1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]oxy}pentanoic acid (Iaa19);

(46)(3S)-5-[(4-benzyl-5-oxo-2,5-dihydro-3-furanyl)oxy-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa20);

(47) (3 S)-5-(isobutyryloxy)-3-({([(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa21);

(48) (3 S)-3-(({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-hexenoicacid (Iaa22);

(49)(3S)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2-pyridinyloxy)pentanoicacid (Iaa23);

(50)(3S)-3-({[5-ethyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2-pyridinyloxy)pentanoicacid (Iaa24);

(51)2-([(3S)-4-carboxy-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-2-oxobutyl]oxy}-1-methylpyridiniumtrifluoromethanesulfonate (Iaa25);

(52)2-{[(3S)-4-carboxy-3-({[5-ethyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-2-oxobutyl]oxy}-1-methylpyridiniumtrifluoromethanesulfonate (Iaa26);

(53) 3-({[3-(5-chloro-1-methyl-1H-indol-2-yl)-5-isopropyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa27);

(54) 3-({[3-(1,5-dimethyl-1H-indol-2-yl)-5-isopropyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa28); and

(55) (3S)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iii-1).

The compound of formula (1) according to the present invention has two(2) asymmetric carbon atoms, and so may exist in its stereoisomeric formincluding diastereomer. Particularly preferred compounds among thestereoisomers are those having the following formula (1a):

and so it is another object of the present invention to provide thecompound of formula (1a) in the stereoisomeric form.

Among the compound of formula (1a), for example, the compound of (3S)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iii-1) may be obtained by a process comprising the steps ofdissolving a mixture of (3 5) and (3R) in methyl t-butyl ether, adding asmall amount of crystalline (3S)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid as a seed material to give a crystal, and recrystallizing thiscrystal from ethyl acetate/n-hexane solvent system.

The most preferred compounds among the compounds of fornulae (1) and(1a) are 5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iii); and (3S)-5-fluoro-3-({[(5R)-5-isopropyl3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iii-1).

The processes for the preparation of the novel isoxazoline derivative offormula (1) showing an inhibitory activity against caspases are depictedin the following Reaction Schemes 1 to 5. However, those illustrated inthe following Reaction Schemes represent only the typical processes usedin the present invention. The manipulation order may be changed with nolimit, and so the processes are not restricted to those explained below.

in which

A, B, and R² are defined as described above, and

P¹ represents simple alkyl chain.

In Reaction Scheme 1 above, an aldehyde derivative (II) is reacted withhydroxylamine-hydrochloride and sodium carbonate in a solvent mixture ofalcohol-water to give an oxime derivative (III) (a mixture of syn andanti oximes). The resulting oxime derivative (III) is treated by NCS(N-chlorosuccinimide) in dimethylformamide solution to give ahydroxamoyl chloride derivative (IV). Thus obtained hydroxamoyl chloridederivative (IV) is reacted with an acrylate derivative (V) to give anisoxazoline derivative (VI), which is then hydrolyzed, if needed, togive a deprotected isoxazoline derivative (VII). If appropriate, theoxime derivative (III), the acrylate derivative (V), and NaOCl may reacttogether in a reaction vessel (in sit-up) to directly give theisoxazoline derivative (VI) (see Preparations 16 and 17).

1(X═COCH₂Z(Z=OR¹¹, OC(═O)R¹², or W) in which A, B, R, R¹, R², R¹¹, R¹²,and W are defined as described above.

In Reaction Scheme 2 above, a carboxylic acid derivative (VII) iscoupled with an aspartic acid derivative (VIII) (see the followingReaction Scheme 3) to give a compound (IX), which is then subjected toDess-Martin periodinane oxidation reaction, and deprotection reaction,if needed, to give the desired compound of formula (1).

The functional group Z in the compound (I) of Reaction Scheme 2 may beformed through several steps as exemplified in the followingPreparations 5 and 7 after the carboxylic acid compound (VII) on theleft side is combined with an aspartic acid (β-t-Bu) methylester.Otherwise, as depicted in the following Reaction Scheme 3, the compound(VIII) already having the desired Z is synthesized first and coupledwith the compound (VII) (Ref.: WO 00/23421). Further, when W is F, theracemic compound may be prepared according to a method known inTetrahedron Letters, 1994, 35(52), 9693-9696.

in which Z is defined as described above.

The acrylate derivative (V) used as a reactant in the above ReactionScheme I may be prepared via two (2) pathways. That is, a compound (XV)can be easily prepared from a known compound (XIV) (Synthesis, 1982, p924) as depicted in the following Reaction Scheme 4, or methyl(ethyl)2-i-propylacrylate, the compound (V), can be prepared fromdimethyl(diethyl)malonate (J. Chemical Society Perkin Trans. 1 1997,1559-1570).

in which P₁ and R² are defined as described above.

The following Reaction Scheme 5 shows a synthetic process for preparinga compound wherein α,β-unsaturated ester or α,β-unsaturated sulfon isintroduced into the P₁ site. According to this, the carboxylic acidderivative (VII) as prepared in Reaction Scheme 1 is reacted with anamino alcohol derivative (XVII) to give a compound (XVI), which is thensubjected to Dess-Martin oxidation and Wittig reactions to give thecompound (I).

in which

R, A, B, R¹, and R² are defined as described above, and

Q represents —CC₂R¹³ or SO₂R¹⁴, wherein R¹³ and R¹⁴ are defined asdescribed above.

The compound of formula (1) according to the present invention has abroad spectrum of inhibitory activity against caspases as demonstratedby the results of the following Experiments, and so has an effect forpreventing inflammation and apoptosis. Thus, the present inventionprovides a composition for inhibiting caspases, specifically atherapeutic composition for preventing inflammation and apoptosis,comprising the compound of formula (1) as an active ingredient togetherwith the pharmaceutically acceptable carrier. Specifically, thecomposition of the present invention has a therapeutic effect fordementia, cerebral stroke, brain impairment due to AIDS, diabetes,gastric ulcer, cerebral injure by hepatitis, hepatitis-induced hepaticdiseases, acute hepatitis, fulminant hepatic failure, liver cirrhosis,sepsis, organ trans-plantation rejection, rheumatic arthritis, orcardiac cell apoptosis due to ischemic cardiac diseases, particularlyspecifically for acute hepatitis, liver cirrhosis, or rheumaticarthritis.

Caspase inhibitor, particularly the compound of formula (1), may beformulated into various pharmaceutical forms for administration purpose.To prepare the pharmaceutical composition according to the presentinvention, an effective amount of the caspase inhibitor, particularlythe compound of formula (1) or its salt, is mixed with apharmaceutically acceptable carrier that may take a wide variety offorms depending on the formulation to be prepared.

The caspase inhibitor compound may be formulated as a parenteralinjection, or percutaneous or oral preparation depending on itsapplication purpose. It is especially advantageous to formulate thecomposition in a unit dosage form for ease of administration anduniformity of dosage.

For the oral preparation, any usual pharmaceutical carrier may be used.For example, water, glycols, oils, alcohols and the like may be used forsuch oral liquid preparations as suspensions, syrups, elixirs andsolutions; or starches, sugars, kaolin, lubricants, binders,disintegrating agents and the like may be used for such solidpreparations as powders, pills, capsules and tablets. Due to their easeof administration, tablets and capsules are the most advantageous dosageunit forms. It is also desirable for tablets and pills to be formulatedinto enteric-coated preparation.

For the parenteral preparation, sterile water is usually used as thecarrier, though other ingredients such as solubility aids may be used.Injections, for example, sterilized aqueous or oily suspension forinjection, can be prepared according to the known procedure usingsuitable dispersing agent, wetting agent, or suspending agent. Solventsthat can be used for preparing injections include water, Ringer's fluid,and isotonic NaCl solution, and also sterilized fixing oil may beconveniently used as the solvent or suspending media. Anynon-stimulative fixing oil including mono-, di-glyceride may be used forthis purpose. Fatty acid such as oleic acid may also be used forinjections.

For the percutaneous administration, the carrier may include apenetration enhancing agent and/or a suitable wetting agent, optionallycombined with suitable additives having no significant skin irritation.Said additives may facilitate the administration through the skin and/ormay assist preparation of a desired composition. These percutaneouspreparations are administered via various manners, e.g., as atransdermal patch, a spot-on, or an ointment.

When the caspase inhibitor, specifically the compound of formula (1), isused for clinical purpose, it is preferably administered to the subjectpatient in an amount ranging from 0.1 to 100 mg per kg of body weight aday. The total daily dosage may be administered once or over severaltimes. However, specific administration dosage for an individual patientcan be varied with specific compound used, body weight, sex, hygieniccondition, or diet of subject patient, time or method of administration,excretion rate, mixing ratio of agent, severity of disease to betreated, etc.

The present invention will be more specifically explained by thefollowing examples. However, it should be understand that these examplesare intended to illustrate the present invention but not in any mannerto limit the scope of the present invention.

Preparation 1

Benzaldehyde Oxime

Benzaldehyde (5.31 g, 50.0 mmol) was dissolved in ethanol (60 ml)-water(30 ml), and hydroxylamine hydrochloride (5.21 g, 1.5 Eq) and anhydroussodium carbonate (Na₂CO₃, 3.97 g, 0.75 Eq) were added thereto at 10° C.When a large amount of solid was formed after about one minute,water-ethanol (1:1, 60 ml) was added, and the mixture was stirred forone hour. Saturated aqueous sodium chloride solution (100 ml) was added,and the mixture was extracted twice with ethyl acetate (300 ml). Theextract was washed with 1.0 N aqueous sodium bicarbonate solution(NaHOCO₃, 100 ml×2), dried (anhydrous Na₂SO₄), and concentrated underreduced pressure to give the title compound (6.06 g, Yield 99%) as awhite powder as a mixture of cis and trans.

¹H-NMR (500 MHz, CDCl₃) δ 8.9(br, 1H), 8.1(s, 1H), 7.5(m, 2H), 7.3(m,3H)

Preparation 2

Benzaldehyde Hydroxamoyl Chloride

The oxime prepared in Preparation 1 (3.00 g, 24.8 mmol) was dissolved indimethyl-formamide (100 ml), and N-chlorosuccinimide (3.47 g, 1.05 Eq)was added. The resulting solution was stirred for one hour in a waterbath of about 40° C., and the volatile solvent was removed bydistillation under reduced pressure. The residue was dissolved in ethylacetate-hexane (1:1, 150 ml), washed with water (100 ml×3), dried(anhydrous Na₂SO₄), and concentrated under reduced pressure to give thetitle compound (3.86 g, Yield 99%). This compound was used in the nextreaction without any further purification.

Preparation 3

Methyl 5-methyl-3-phenyl-4,5-dihydro-5-isoxazolecarboxylate (VIa)

The hydroxamoyl chloride prepared in Preparation 2 (3.86 g, 24.8 mmol)and methyl methacrylate (4.0 ml , 37.2 mmol, 1.5 Eq) were dissolved indry diethylether (120 ml) under nitrogen atmosphere, the mixture waskept at −78° C., and triethylamine (6.9 ml, 2.0 Eq) was added. Themixture was stirred overnight while it was slowly warmed to roomtemperature. Water (100 ml) was added, and then the mixture wasextracted with ethyl acetate (100 ml×2), washed with water (100 ml),dried (anhydrous Na₂SO₄), and concentrated under reduced pressure. Theresidue was purified by column chromatography (15% ethyl acetate-hexane)to give the title compound (5.34 g, Yield 98%), which was thenidentified by ¹H-NMR to be 1:1 mixture of diastereomers.

¹H-NMR(400 MHz, CDCl₃) δ 6.7(m, 2H,), 6.5(m, 3H), 3.0(d, J=16.7 Hz, 1H),2.9(s, 3H), 2.3(d, J=16.7 Hz, 1H), 0.8(s, 3H)

Preparation 4

5-Methyl-3-phenyl-4,5-dihydro-5-isoxazolecarboxylic acid (VIIa)

The compound prepared in Preparation 3 (VIa, 5.34 g) was dissolved indistilled tetrahydrofuran (120 ml), and 1N aqueous sodium hydroxidesolution (36.5 ml, 1.5 Eq) was added. After about four hours, themixture was neutralized by 1N aqueous hydrochloric acid solution anddistilled under reduced pressure to remove most of tetrahydrofuran. Theresidue was dissolved in excess ethyl acetate (>700 ml), washed withwater, dried (anhydrous Na₂SO₄), and concentrated under reduced pressureto give the title compound (4.77 g, Yield 95%) as a white powder. Thiscompound was used in the next reaction without any further purification.

Preparation 5

(3S)-3-{[(benzyloxy)carbonyl]amino}-5-(t-butoxy)-2-hydroxy-5-oxopentyl2,6-dichlorobenzoate (XIIa)

To N-benzyloxycarbonyl-β-t-butylaspartic acid (5.03 g, 15.6 mmol) andNMM (1.90 ml, 17.1 mmol) was added dry tetrahydrofuran (60 ml) undernitrogen atmosphere, the mixture was kept −15° C., and isobutylchloroformate (2.12 ml, 16.3 mmol) was added thereto. The mixture wasstirred for about 20 minutes. To the reaction mixture being kept at 0°C. was added diazomethane-ether solution (synthesized from 2.0 Eq of1-methyl-3-nitro-1-nitroso-guanidine, 60 ml) to prepare diazoketonederivative (˜30 minutes). 30% HBr/AcOH (6.42 ml, 2.0 Eq) was addedthereto at 0° C. The resulting mixture was stirred (30-60 minutes),extracted with ethyl acetate, washed twice with saturated aqueous sodiumbicarbonate solution and once with aqueous sodium chloride solution,dried (anhydrous Na₂SO₄), and concentrated under reduced pressure togive bromomethyl ketone derivative (X, 6.4 g).

Thus obtained bromomethyl ketone derivative (X, 4.36 g) and2,6-dichlorobenzoic acid (2.28 g, 1.1 Eq) were dissolved indimethylformamide (18 ml), KF (1.58 g, 2.5 Eq) was added, and themixture was stirred for two hours to give 2,6-dichlorobenzoyloxymethylketone derivative (XIa). This compound was dissolved in methanol (20 ml)and reacted by adding NaBH₄(412 mg)-methanol solution (40 ml)(−10°C.˜room temperature, two hours). The reaction was stopped by aceticacid, and the reaction solution was distilled under reduced pressure toremove methanol. The residue was extracted with ethyl acetate (50 ml×2),washed with water and aqueous sodium chloride solution, dried (anhydrousNa₂SO₄), concentrated under reduced pressure, and separated-purified bycolumn chromatography (ethyl acetate-hexane, 1:5) to give the titlecompound (4.80 g, Yield 86%) in a diastereomeric form.

¹H-NMR (400 MHz, CDCl) δ 7.3-7.2(m, 8H), 5.9(m, 1H), 5.2(m, 4H), 4.7(m,1H), 2.9(m, 1H), 2.7(m, 1H), 1.4(s, 9H)

Preparation 6

(3S)-3-amino-5-(t-butoxy)-2-hydroxy-5-oxopentyl 2,6-dichlorobenzoate(VIIIa)

The benzyloxycarbonyl group of the compound prepared in Preparation 5was removed (Pd/C) under hydrogen balloon for 40 minutes to give thetitle compound (Yield 100%).

¹H-NMR (400 MHz, DMSO-d₆) δ 8.2(br, 2H), 7.6-7.5(m, 3H), 6.1(m, 1H),4.4-3.9(m, 3H), 3.0-2.6(m, 2H), 1.4(s, 9H)

EXAMPLE 1(3-5-(t-butoxy)-3-{[(5-methyl-3-phenyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-2,5-dioxopentyl2,6-dichlorobenzoate (Ia)

A mixture of the carboxylic acid derivative prepared in Preparation 4(VIIa, 300 mg, 1.46 mmol), the amino alcohol derivative prepared inPreparation 6 (VIIIa, 667 mg, 1.1 Eq) and HATU (722 mg, 1.3 Eq) wascooled to 0° C., triethylamine (0.82 ml, 4.0 Eq) was added in a solventof DMF(5 ml), and the resulting mixture was reacted for 5 hours. Thesolvent was distilled under reduced pressure, and the residue wasextracted with ethyl acetate (200 ml×2), washed with water, aqueoussodium bicarbonate solution, and aqueous sodium chloride solution, dried(anhydrous Na₂SO₄), concentrated under reduced pressure, and purified bycolumn chromatography to give Compound (IXa) (810 mg, Yield 98%). Tothis compound and Dess-Martin reagent (1.70 g, 3.0 Eq) was added drydichloromethane (25 ml), and the mixture was stirred for one hour atroom temperature. The reaction was stopped by isopropyl alcohol (3 ml).The solid was removed by filtration under reduced pressure throughCelite, and the filtrate was extracted with ethyl acetate (20 ml×2),washed with water, saturated aqueous sodium bicarbonate solution andaqueous sodium chloride solution, dried (anhydrous Na₂SO₄), concentratedunder reduced pressure, and preliminarily purified by columnchromatography (36% ethyl acetate-hexane) to give the diastereomerictitle compound (780 mg).

¹H-NMR(400 MHz, CDCl₃) δ 7.8(m, NH, 1H), 7.6(m, 2H), 7.3(m, 3H), 7.2(m,3H), 5.1-5.0(m, 2H), 4.8(m, 1H), 3.8(m, 1H), 3.2(m, 1H), 2.9-2.8(m, 2),1.7(s, 3H), 1.4(s, 9H)

EXAMPLE 2(3S)-5-[(2,6-dichlorobenzoyl)oxy]-3-{[(5-methyl-3-phenyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-4-oxopentanoicacid (Iaa)

The compound prepared in Example 1 (44 mg) was dissolved indichloromethane (2 ml), and trifluoroacetic acid (1 ml) was added at 0°C. The mixture was stirred for two hours while it was slowly warmed toroom temperature, and then concentrated under reduced pressure to givethe title compound (Iaa) in a stoichiometric amount.

¹H-NMR(500 MHz, CDCl₃) δ 8.1(m, 1H), 7.5((m, 2H), 7.3(m, 3H), 7.2(m,3H), 5.1-4.9(m, 2H), 4.9-4.8(m, 1H), 3.8(m, 1H), 3.2(m, 1H), 1.7(m, 3H)

Preparation 7

t-Butyl (3S)-3-amino-4-hydroxy-5-phenoxypentanoate (VIIIb)

To N-benzyloxycarbonyl-β-t-butyl-aspartic acid (10.0 g, 31.0 mmol) andNMM (3.75 ml, 1.1 Eq) was added dry tetrahydrofuran (120 ml) undernitrogen atmosphere, and the mixture was kept at −15° C. Isobutylchloroformate (4.22 ml, 1.05 Eq) was added thereto, and the mixture wasstirred for about 20 minutes. The reactants were kept at 0° C., whichwas then mixed with diazomethane-ether solution (synthesized from 2.0 Eqof 1-methyl-3-nitro-1-nitrosoguanidine, 60 ml) to prepare diazoketonederivative (˜30 minutes). 30% HBr/AcOH (12.83 ml, 2.0 Eq) was addedthereto to prepare bromomethyl ketone derivative (30-60 minutes). Theproduct was extracted with ethyl acetate, washed twice with saturatedaqueous sodium bicarbonate solution and aqueous sodium chloridesolution, dried (anhydrous Na₂SO₄), and concentrated under reducedpressure to give bromomethyl ketone derivative (12.9 g) in astoichiometric amount.

Thus obtained bromomethyl ketone derivative (X, 12.9 g, 31.0 mmol) andphenol (3.23 g, 1.2 Eq) were dissolved in dimethylformamide (30 ml),KF(4.53 g, 2.5 Eq) was added, and the mixture was stirred for two hoursto give phenoxymethyl ketone derivative (XIb). This compound wasdissolved in methanol (40 mg)-THF (100 ml) and was reacted by addingNaBH₄(2.35 g)-methanol solution (40 ml)(−10° C.—room temperature, twohours). The reaction was stopped by acetic acid, and the reactionmixture was distilled under reduced pressure to remove methanol. Theresidue was extracted with ethyl acetate (50 ml×2), washed with waterand aqueous sodium chloride solution, dried (anhydrous Na₂SO₄),concentrated under reduced pressure, and separated-purified by columnchromatography (ethyl acetate-hexane, 1:7) to give a compound (XIIb,6.50 g, Yield 50%) in a diastereomeric form of which amino group isprotected by benzyloxycarbonyl.

¹H-NMR(400 MHz, CDCl₃) δ 7.3-7.2(m, 8H), 5.9(m, 1H), 5.2(m, 4H), 4.7(m,1H), 2.9(m, 1H), 2.7(m, 1H), 1.4(s, 9H)

The benzyloxycarbonyl group of the compound obtained above was removed(Pd/C) under hydrogen balloon to give the title compound (4.16 g, Yield95%).

¹H-NMR(400 MHz, DMSO-d₆) δ 8.1(br, 2H), 7.3(m, 5H), 5.6(m, 1H), 4.1-4.0(m, 3H), 2.6(m, 2H), 1.4(s, 9H)

EXAMPLE 3 t-butyl(3S)-3-{[(5-methyl-3-phenyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-4-oxo-5-phenoxypentanoate(Ib)

A mixture of the carboxylic acid derivative prepared in Preparation 4(VIIa, 273 mg, 1.33 mmol), the amino alcohol derivative prepared inPreparation 7 (VIIIb, 412 mg, 1.1 Eq) and HATU (657 mg, 1.3 Eq) wascooled to 0° C., triethylamine (0.74 ml, 4.0 Eq) was added in a solventof DMF(5 ml), and the resulting mixture was reacted for 5 hours. Thesolvent was distilled under reduced pressure, and the residue wasextracted with ethyl acetate (200 ml×2), washed with water, aqueoussodium bicarbonate solution, and aqueous sodium chloride solution, dried(anhydrous Na₂SO₄), concentrated under reduced pressure, and purified bycolumn chromatography (30-40% EA/Hex) to give Compound (IXb) (545 mg,Yield 88%). To this compound and Dess-Martin reagent (1.43 g, 3.0 Eq)was added dry dichloromethane (25 ml), and the mixture was stirred forone hour at room temperature. The reaction was stopped by isopropylalcohol (2 ml). The solid was removed by filtration under reducedpressure through Celite, and the filtrate was extracted with ethylacetate (20 ml×2), washed with water, saturated aqueous sodiumbicarbonate solution and aqueous sodium chloride solution, dried(anhydrous Na₂SO₄), concentrated under reduced pressure, andpreliminarily purified by column chromatography (eluent: 25% ethylacetate-hexane) to give the diastereomeric title compound (540 mg, Yield99%).

¹H-NMR(400 MHz, CDCl₃) δ 7.8(NH, 1H), 7.5(m, 2H), 7.3(m, 3H), 7.3(m,1H), 7.1(m, 1H), 6.8(m, 2H), 6.7(m, 1H), 4.9(s, 1H), 4.7-4.6(m, 2H),3.7(d, J=17 Hz, 1H), 3.2(d, J=17 Hz, 1H), 2.9(m, 1H), 2.8(m, 1H), 1.7(s,3H), 1.4(s, 9H)

EXAMPLE 4(3S)-3-{[(5-methyl-3-phenyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-4-oxo-5-phenoxypentanoicacid (Ibb)

The compound prepared in Example 3 (530 mg, 1.136 mmol) was dissolved indichloromethane (6 ml), and trifluoroacetic acid (3 ml) was added at 0°C. The mixture was stirred for two hours while it was slowly warmed toroom temperature, and concentrated under reduced pressure to give thetitle compound (Ibb) (465 mg) with a stoichiometric yield.

¹H-NMR(400 MHz, CDCl₃) δ 7.5(m, 2H), 7.3(m, 3H), 7.1(NH, 1H), 6.7(m,5H), 5.0-4.6(m, 2H), 4.2(m, 1H), 3.8(m, 1H), 3.2(m, 1H), 3.0-2.7(m, 2H),1.6(s, 3H)

Preparation 8

Ethyl 2-ethylacrylate (XVb)

To CuCN (26.9 g, dried in advance under vacuum) was added under nitrogenatmosphere about 500 ml of dry tetrahydrofuran. The mixture was kept at−78° C., and 100 ml of methyl magnesium bromide (3.0M diethylethersolution) was slowly added thereto under mechanical stirring. The thickmixture was stirred for about 30 minutes at −78° C. Ethyl2-bromomethylacrylate (28.9 g, 150 mmol, Synthetic method: Villieras, J.and Rambaud, M. Synthesis, 1982, 914) dissolved in about 30 ml of drytetrahydrofuran was slowly added thereto. The temperature of thereaction mixture was slowly raised to room temperature over two hours.The reaction was completed by slowly adding saturated ammonium chloridesolution (˜50 ml). The reaction mixture was filtered through Celite toremove the precipitates, and then washed with diethylether. The organiclayer was washed with water and saturated sodium bicarbonate solution(300 ml×2), dried (anhydrous Na₂SO₄), and concentrated under reducedpressure to give 26.7 g (stoichiometric yield) of a transparent liquid.This liquid was identified by ¹H-NMR (500 MHz, CDCl₃) and was confirmedto be the title compound having about 75% of w/w purity.

¹H-NMR(500 MHz, CDCl₃) δ 6.12(1H, s), 5.50(1H, s), 4.20(2H, q, J=7.3Hz), 2.31(2H, qt), 1.28(3H, t, J=7.3 Hz), 1.07(3H, t, J=7.8 Hz)

Preparation 9

Ethyl 5-ethyl-3-phenyl-4,5-dihydro-5-isoxazolecarboxylate (VIb)

The compound of Preparation 8 (XVa) was reacted according to the sameprocedure as Preparation 3 to give the title compound.

¹H-NMR(400 MHz, CDCl₃) δ 7.6(m, 2H), 7.4-7.3(m, 3H), 4.3-4.2(m, 2H),4.0(d, J=17.2 Hz, 1H), 3.4(d, J=17.2 Hz, 1H), 2.1-2.0(m, 2H), 1.3(t,3H), 1.0(t, 3H)

Preparation 10

t-Butyl (3S)-3-amino-4-hydroxy-5-(2,3,5,6-tetrafluorophenoxy)pentanoate(VIIIc)

2,3,5,6-Tetrafluorophenol was reacted according to the same procedure asPreparations 5 and 6 to give the title compound in a total yield of 72%.

¹H-NMR(400 MHz, DMSO-d₆) δ 8.2(br, 2H), 7.6-7.5(m, 1H), 5.9(m, 1H),4.3-4.1(m, 3H), 3.6(m, 1H), 2.7(m, 1H), 1.4(s, 9H)

EXAMPLE 5 t-Butyl(3S)-3-{[(5-ethyl-3-phenyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoate(Ic)

The compounds of Preparations 9 and 10 were reacted according to thesame procedure as Preparation 4 and Example 1 to give the titlecompound.

¹H-NMR(400 MHz, CDCl₃) δ 7.6(dd, J=3.6, 1.6 Hz, 1H), 7.6(m, 2H),7.4-7.3(m, 3H), 6.7(m, 1H), 5.1-4.9(m, 2H), 4.9-4.8(m, 1H), 3.7(dd,J=17.6, 17.2 Hz, 1H), 3.3(1H, d, J=17.2 Hz), 3.0-2.8(m, 1H), 2.8-2.7(m,1H), 2.1(m, 1H), 1.9(m, 1H), 1.4-1.3(two s, 9H), 1.0(m, 3H)

EXAMPLE 6(3S)-3-{[(5-ethyl-3-phenyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Icc)

The compound of Example 5 was reacted according to the same procedure asExample 2 to give the title compound.

¹H-NMR(400 MHz, DMSO-d₆) δ 7.6(br, 1H), 7.6-7.5(m, 2H), 7.4-7.3(m, 3H),6.8-6.7(m, 1H), 4.9-4.8(m, 1H), 4.5(br, 2H), 3.7(d, J=16 Hz, 1H), 3.3(d,J=16 Hz, 1H), 3.3-3.0(m, 1H), 2.8-2.7(m, 1H), 2.1(m, 1H), 2.0-1.9(m,1H), 1.0(m, 3H))

MS [M+H]⁺ 497

Preparation 11

Ethyl 5-ethyl-3-(1-naphthyl)-4,5-dihydro-5-isoxazolecarboxylate (VIc)

1-Naphthaldehyde and ethyl 2-ethylacrylate were reacted according to thesame procedure as Preparations 1, 2, and 3 to give the title compound.

¹H-NMR(400 MHz, CDCl₃) δ 8.9(d, J=8.8 Hz, 1H), 7.9-7.8(m, 2H),7.6-7.4(m, 4H), 4.3-4.2(m, 2H), 4.0(d, J=17.2 Hz, 1H), 3.4(d, J=17.2 Hz,1H), 2.1-2.0(m, 2H), 1.3(t, 3H), 1.0(t, 3H)

EXAMPLE 7 t-Butyl(3S)-3-({[5-ethyl-3-(1-naphthyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoate(Id)

The compounds of Preparations 11 and 10 were reacted according to thesame procedure as Preparation 4 and Example 1 to give the titlecompound.

¹H-NMR(400 MHz, CDCl₃) δ 8.9(m, 1H), 7.9-7.8(m, 3H), 7.6-7.4(m, 4H),6.5-6.9(m, 1H), 5.1-4.9(m, 2H), 4.9(m, 1H), 3.9(dd, 1H), 3.5(d, J=17.2Hz, 1H), 3.0-2.9(m, 1H), 2.8(m, 1H), 2.2(m, 1H), 2.0(m, 1H), 1.4-1.3(twos, 9H), 1.1(m, 3H)

EXAMPLE 8(3S)-3-({[5-ethyl-3-(1-naphthyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Idd)

The compound of Example 7 was reacted according to the same procedure asExample 2 to give the title compound.

¹H-NMR(400 MHz, CDCl₃) δ 8.8(m, 1H), 7.9-7.7(m, 3H), 7.6-7.4(m, 4H),6.7(m, 1H), 4.9(m, 1H), 4.5(br, 2H), 3.9(d, J=17.2 Hz, 1H), 3.5(d,J=17.2 Hz, 1H), 3.1-2.9(m, 1H), 2.8-2.7(m, 1H), 2.2(m, 1H), 2.0(m, 1H),1.0(m, 3H)

MS [M+MeOH+Na]⁺ 601

Preparation 12

Ethyl 5-ethyl-3-(2-naphthyl)-4,5-dihydro-5-isoxazolecarboxylate (VId)

2-Naphthaldehyde and ethyl 2-ethylacrylate were reacted according to thesame procedure as Preparations 1, 2, and 3 to give the title compound.

¹H-NMR(500 MHz, CDCl₃) δ 8.7(d, J=8.8Hz, 1H), 7.8-7.7(m, 2H), 7.5-7.3(m,4H), 4.3-4.2(m, 2H), 4.0(d, J=17.2 Hz, 1H), 3.4(d, J=17.2 Hz, 1H),2.1-2.0(m, 2H) 1.3(t, 3H), 1.0(t, 3H)

EXAMPLE 9 t-Butyl(3S)-3-({[5-ethyl-3-(2-naphthyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoate(Ie)

The compounds of Preparations 12 and 10 were reacted according to thesame procedure as Preparation 4 and Example 1 to give the titlecompound.

¹H-NMR(400 MHz, CDCl₃) δ 7.8-7.7(m, 6H), 7.5(m, 2H), 6.7 & 6.5 (m, 1H),5.1-4.9(m, 2H), 4.9-4.8(m, 2H), 3.8(dd, J=16 Hz, 1H), 3.4(d, J=16 Hz,1H), 3.0(m, 1H), 2.8(m, 1H), 2.8-2.7(m, 1H), 2.2-2.1(m, 1H), 2.0(m, 1H),1.4-1.3 (two s, 9H), 1.1-1.0(m, 3H)

EXAMPLE 10(3S)-3-({[5-ethyl-3-(2-naphthyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Iee)

The compound of Example 9 was deprotected according to the sameprocedure as Example 2, and the isomers were resolved by prep-TLC togive the following two compounds.

Weakly polar diastereomer:

¹H-NMR(400 MHz, DMSO-d₆) δ 12.4(br, 1H), 8.7(br, 1H), 8.1(s, 1H),7.9-8.0(m, 3H), 7.8-7.9(m, 1H), 7.5-7.6(m, 3H), 5.2(br, 2H), 4.7(br,1H), 3.8(d, J=17.2 Hz, 1H), 3.5(d, J=17.6 Hz, 1H), 2.7(m, 1H), 2.5(m,1H), 2.0(m, 1H), 1.9(m, 1H), 0.9-0.8(m, 3H)

MS [M+H]⁺ 547

Highly polar diastereomer:

¹H-NMR(400 MHz, DMSO-d₆) δ 8.6(br, 1H), 8.2(s, 1H), 8.0-7.9(m, 3H),7.9(m, 1H), 7.6(m, 2H), 7.4(m, 1H), 5.0(br, 2H), 4.8(m, 1H), 3.9(d,J=17.6 Hz, 1H), 3.6(d, J=17.6 Hz, 1H), 2.9-2.7(m, 2H), 2.1-2.0(m, 1H),2.0-1.9(m, 1H), 0.9(m, 3H)

Preparation 13

Ethyl 5-ethyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolecarboxylate(VIe)

1-Isoquinolinealdehyde and ethyl 2-ethylacrylate were reacted accordingto the same procedure as Preparations 1, 2 and 3. The title compound inan active chiral isomeric form was separated by Chiral OD HPLC (DaicelChemical Industries, 2.00 cm×25 cm, OD00CJ-1C005, 3% i-PrOH in Hexane,220 nm), which was then used in the next reaction.

¹H-NMR(400 MHz, CDCl₃) δ 9.25(m, 1H), 8.55(d, 1H), 7.85(d, 1H),7.74-7.65(m, 3H), 4.29(m, 2H), 4.13(d, 1H), 3.71(d, 1H), 2.11(m, 2H),1.33(t, 3H), 1.06(t, 3H)

EXAMPLE 11

t-Butyl(3S)-3-({[5-ethyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoate(If)

The compounds of Preparations 13 and 10 were reacted according to thesame procedure as Preparation 4 and Example 1 to give the titlecompound.

¹H-NMR(400 MHz, CDCl₃) δ 9.14(m, 1H), 8.53(m, 1H), 7.91-7.85(m, 2H),7.74-7.64(m, 3H), 6.73-6.62(m, 1H), 5.30-4.91(m, 3H), 4.09(two d, 1H),3.72(two d, 1H), 3.04-2.76(m, 2H), 2.24(m, 1H), 2.04(m, 1H), 1.45 &1.35(two s, 9H), 1.08(two t, 3H)

EXAMPLE 12(3S)-3-({[5-ethyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Iff)

The compound of Example 11 was reacted according to the same procedureas Example 2 to give the title compound.

¹H-NMR(400 MHz, DMSO-d₆) δ 9.02(m, 1H), 8.76(m, 1H), 8.59(m, 1H),8.08-7.73(m, 4H), 7.61-7.32(m, 1H), 5.19-5.10(m, 2H), 4.93-4.65(m, 1H),3.91 (two d, 1H), 3.68(two d, 1H), 2.91-2.52(m, 2H), 2.10-1.94(m, 2H),0.94(two t, 3H)

Mass: M+H 548

EXAMPLE 13 t-Butyl3-({[5-ethyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoate(Ig)

The compound of Preparation 13 and t-butyl3-amino-5-fluoro-4-hydroxypentanoate were reacted according to the sameprocedure as Preparation 4 and Example 1 to give the title compound.

¹H-NMR(400 MHz, CDCl₃) δ 9.15(m, 1H), 8.55(d, 1H), 7.87-7.66(m, 5H),5.22-4.89(m, 3H), 4.12(two d, 1H), 3.72(two d, 1H), 3.05-2.75(m, 2H),2.22 (m, 1H), 2.04(m, 1H), 1.45 & 1.34(two s, 9H), 1.07(two t, 3H)

EXAMPLE 143-({[5-Ethyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Igg)

The compound of Example 13 was reacted according to the same procedureas Example 2 to give the title compound.

¹H-NMR(400 MHz, DMSO-d₆) δ 9.03(m, 1H), 8.67-8.59(m, 2H), 8.08(d, 1H),7.97-7.78(m, 3H), 5.26-5.07(m, 2H), 4.75(m, 1H), 3.94(two d, 1H),3.67(two d, 1H), 2.88-2.58(m, 2H), 2.07-1.94(m, 2H), 0.96(two t, 3H)

Mass: M+H 402

EXAMPLE 15 Ethyl3-({[5-ethyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoate(Ihh)

The compound of Example 14 was reacted according to a method known inTetrahedron Letters, 1994, 35(52), 9693-9696 to give the title compound.

¹H-NMR(400 MHz, CDCl₃) δ 9.15(m, 1H), 8.55(d, 1H), 7.89-7.67(m, 5H),5.23-4.94(m, 3H), 4.18(m, 2H), 4.11(two d, 1H), 3.72(two d, 1H),3.08-2.82 (m, 2H), 2.22(m, 1H), 2.05(m, 1H), 1.29-1.04(m, 6H)

Preparation 14

Methyl5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolecarboxylate) andresolution (VIf)

1-Isoquinolinealdehyde and methyl 2-isopropylacrylate were reactedaccording to the same procedure as Preparations 1, 2 and 3 to give thetitle compound. From thus obtained compound, (5R)-active chiral isomer(9.7 min-11.7 min) was separated by prep-HPLC using chiral OD column(Daicel Chemical Industries, 2.00 cm×25 cm, OD00CJ-1C005, 5% i-PrOH inHexane, 14 ml/min, 220 nm) (the other (5S)-isomer was eluted during theretention time of 15.3-20.1 min), which was then used in the nextreaction (see Preparation 23 for the resolution after hydrolysis)

¹H-NMR(500 MHz, CDCl₃) δ 9.24(m, 1H), 8.55(d, 1H), 7.85(d, 1H), 7.72(t,3H), 7.67(m, 2H), 4.11(d, 1H), 3.83(s, 3H), 3.74(d, 1H), 2.50(septet,1H), 1.07(d, 3H), 1.02(d, 3H)

EXAMPLE 16 t-Butyl5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoate (Ih)

The compound of Preparation 14 and t-butyl3-amino-5-fluoro-4-hydroxypentanoate were reacted according to the sameprocedure as Preparation 4 and Example 1 to give the title compound.

¹H-NMR(500 MHz, CDCl₃) δ 9.14(m, 1H), 8.55(d, 1H), 7.87-7.65(m, 5H),5.23-4.93(m, 2H), 4.90(m, 1H), 4.05-4.00(two d, 1H), 3.84-3.79(two, 1H),3.06-2.74(m, 2H), 2.40(m, 1H), 1.45 & 1.34(two s, 9H), 1.12-1.07(m, 6H)

EXAMPLE 175-Fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iii)

The compound of Example 16 was reacted according to the same procedureas Example 2 to give the title compound (the compound of (5S)-isomer,Iii-u, was obtained according to the same procedure).

¹H-NMR(400 MHz, DMSO-d₆) δ 9.00(m, 1H), 8.63-8.48(m, 2H), 8.08(d, 1H),7.97(m, 1H) 7.87-7.76(m, 2H), 5.31-4.82(br, 2H), 4.74(m, 1H), 3.91(twod, 1H), 3.73(two d, 1H), 2.88-2.61(m, 2H), 2.33(m, 1H), 0.98(m, 6H)

Mass: M+H 416

Preparation 15

Ethyl 5-ethyl-3-(4-quinolinyl)-4,5-dihydro-5-isoxazolecarboxylate (VIg)

4-Quinoinealdehyde and ethyl 2-ethylacrylate were reacted according tothe same procedure as Preparations 1, 2, and 3 to give the titlecompound.

¹H-NMR(400 MHz, CDC₃) δ 8.95(d, 1H), 8.89(m, 1H), 8.11(d, 1H), 7.83(m,1H), 7.71(m, 1H), 7.65(d, 1H), 4.27(m, 2H), 4.01(d, 1H), 3.67(d, 1H),2.10 (m, 2H), 1.31(t, 3H), 1.02(t, 3H)

EXAMPLE 18 t-Butyl3-({[5-ethyl-3-(4-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoate(Ii)

The compound of Preparation 15 and t-butyl3-amino-5-fluoro-4-hydroxypentanoate were reacted according to the sameprocedure as Preparation 4 and Example 1 to give the title compound.

¹H-NMR(400 MHz, CDC₃) δ 8.97(d, 1H), 8.89(m, 1H), 8.17(d, 1H),7.87-7.64(m, 3H), 7.36(d, 1H), 5.21-4.93(m, 3H), 3.93(two d, 1H),3.48(two d, 1H), 3.05-2.78(m, 2H), 2.24(m, 1H), 2.03(m, 1H), 1.46 &1.33(two s, 9H), 1.06(two t, 3H)

EXAMPLE 193-({[5-Ethyl-3-(4-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Ijj)

The compound of Example 18 was reacted according to the same procedureas Example 2 to give the title compound.

¹H-NMR(400 MHz, DMSO-d₆) δ 9.16-9.10(m, 2H), 8.25-7.86(m, 5H), 5.20(br,2H), 4.75(m, 1H), 4.00-3.89(two d, 1H), 3.73(two d, 1H), 2.87-2.71(m,2H), 2.26-2.05(m, 2H), 1.07(two t, 3H)

Mass: M+H 402

Preparation 16

Ethyl 3-(benzothiophen-2-yl)-5-ethyl-4,5-dihydro-5-isoxazolecarboxylate(VIh)

Benzothiophen-2-aldehyde oxime derivative (400 mg, 2.26 mmol) preparedaccording to the same procedure as Preparation 1 was dissolved in THF(25 ml), and the mixture was kept at 0° C. Ethyl 2-ethylacrylate (434mg, 1.5 Eq) and triethylamine (7 drops) were dissolved in THF (5 ml),thus obtained solution was slowly added to the above, and sodiumhypochlorite solution (6.0 ml) was further added. The reaction solutionwas warmed to room temperature and stirred for 4 hours. Water (20 ml)was added, and the mixture was extracted with ethyl acetate (40 ml×2),washed with aqueous sodium chloride solution (20 ml), dried overmagnesium sulfate, filtered, and concentrated under reduced pressure.The residue was purified by column chromatography (20% ethylacetate-hexane) to give the title compound (121 mg, Yield 18%).

¹H-NMR(400 MHz, CDCl₃) δ 7.82-7.75(m, 2H), 7.40-7.32(m, 3H), 4.28(m,2H), 3.90(d, 1H), 3.31(d, 1H), 2.07(m, 2H), 1.33(t, 3H), 1.01(t, 3H)

EXAMPLE 20 t-Butyl3-({[3-(benzothiophen-2-yl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoate(Ij)

The compound of Preparation 16 and t-butyl3-amino-5-fluoro-4-hydroxypentanoate were reacted according to the sameprocedure as Preparation 4 and Example 1 to give the title compound.

¹H-NMR(400 MHz, CDCl₃) δ 7.83-7.68(m, 3H), 7.42-7.35(m, 3H),5.21-4.88(m, 3H), 3.80(two d, 1H), 3.38(two d, 1H), 3.03-2.78(m, 2H),2.17(m, 1H), 2.01(m, 1H), 1.46 & 1.38(two s, 9H), 1.03(two t, 3H)

EXAMPLE 213-({[3-(benzothiophen-2-yl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid(3-({[3-(benzothiophen-2-yl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid) (Ikk)

The compound of Example 20 was reacted according to the same procedureas Example 2 to give the title compound.

¹H-NMR(400 MHz, DMSO-d₆) δ 8.63(m, 1H), 8.11-7.79(m, 3H), 7.47-7.36(m,2H), 5.24-5.06(m, 2H), 4.73(m, 1H), 3.79(two d, 1H), 3.53(two d, 1H),2.83-2.58(m, 2H), 2.03-1.86(m, 2H), 0.91(two t, 3H)

Mass: M+H 407

Preparation 17

Ethyl3-(1,3-dimethyl-1H-indol-2-yl)-5-ethyl-4,5-dihydro-5-isoxazole-carboxylate(VIi)

1,3-Dimethyl-1H-indol-2-aldehyde oxime derivative (356 mg, 2.07 mmol)was dissolved in dichloromethane (20 ml), and the mixture was kept at 0°C. Ethyl 2-ethylacrylate (345 mg, 1.3 Eq) and triethylamine (6 drops)were dissolved in dichloromethane (5 ml), thus obtained solution wasslowly added to the above, and sodium hypochlorite solution (5.5 ml) wasfurther added. The reaction solution was warmed to room temperature andstirred for 4 hours. Water (20 ml) was added, and the mixture wasextracted with dichloromethane (40 ml×2), washed with aqueous sodiumchloride solution (20 ml), dried over magnesium sulfate, filtered, andconcentrated under reduced pressure. The residue was purified by columnchromatography (15% ethyl acetate-hexane) to give the title compound(230 mg, Yield 35%).

¹H-NMR(400 MHz, CDCl₃) δ 7.60(d, 1H), 7.31(m, 2H), 7.12(m, 1H), 4.29(m,2H), 3.98(d, 1H), 3.94(s, 3H), 3.40(d, 1H), 2.47(s, 3H), 2.08(m, 2H),1.34(t, 3H), 1.04(t, 3H)

Mass: M+H 315

EXAMPLE 22 t-Butyl(3S)-3-({[3-(1,3-dimethyl-1H-indol-2-yl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoate(Ik)

The compounds of Preparations 17 and 10 were reacted according to thesame procedure as Preparation 4 and Example 1 to give the titlecompound.

¹H-NMR(400 MHz, CDCl₃) δ 7.85-7.79(m, 1H), 7.57(m, 1H), 7.37-7.26(m,2H), 7.13(m, 1H), 6.75-6.49(m, 1H), 5.12-4.90(m,3H), 3.95(two d,1H),3.89 (two s,3H), 3.45(two d,1H), 3.03-2.79(m, 2H), 2.44(two s, 3H),2.22(m, 1H), 2.01(m, 1H), 1.58 & 1.37(two s, 9H), 1.05(two t, 3H)

EXAMPLE 23(3S)-3-({[3-(1,3-dimethyl-1H-indol-2-yl)-5-ethyl-4,5-dihydro-5-isoxazol]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Ill)

The compound of Example 22 was reacted according to the same procedureas Example 2 to give the title compound.

¹H-NMR(400 MHz, DMSO-d₆) δ 8.67(br, 1H), 7.61-7.43(m, 3H), 7.27(m, 1H),7.08(m, 1H), 5.20(br, 2H), 4.83(m, 1H), 3.84(two s,3H), 3.76(two d, 1H),3.59(two d, 1H), 2.91-2.60(m, 2H), 2.50(two s, 3H), ^(2.07)-1.91(m, 2H),0.95(two t, 3H)

EXAMPLE 24 t-Butyl3-({[3-(1,3-dimethyl-1H-indol-2-yl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoate(Il)

The compound of Preparation 17 and t-butyl3-amino-5-fluoro-4-hydroxypentanoate were reacted according to the sameprocedure as Preparation 4 and Example 1 to give the title compound.

¹H-NMR(400 MHz, CDCl₃) δ 7.79(m, 1H), 7.60(m, 1H), 7.32(m, 2H), 7.13(m,1H), 5.20-4.90(m,3H), 3.97-3.89(m, 4H), 3.46(two d, 1H), 3.03-2.77(m,2H), 2.21(m, 1H), 2.01(m, 1H), 1.46 & 1.38(two s, 9H), 1.05(two t, 3H)

EXAMPLE 253-({[3-(1,3-Dimethyl-1H-indol-2-yl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Imm)

The compound of Example 24 was reacted according to the same procedureas Example 2 to give the title compound.

¹H-NMR(400 MHz, DMSO-d₆) δ 8.68(br, 1H), 7.60(m, 1H), 7.45(m, 1H),7.26(m, 1H), 7.07(m, 1H), 5.20(br, 2H), 4.81(m, 1H), 3.84(two s, 3H),3.76 (two d, 1H), 3.59(two d, 1H), 2.94-2.59(m, 2H), 2.38(two s, 3H),2.07-1.91 (m, 2H), 0.95(two t, 3H)

Mass: M+H 418

Preparation 18

Ethyl 5-ethyl-3-(1-naphthylmethyl)-4,5-dihydro-5-isoxazolecarboxylate(VIj)

1-Naphthylacetaldehyde and ethyl 2-ethylacrylate were reacted accordingto the same procedure as Preparations 1, 2, and 3 to give the titlecompound.

¹H-NMR(500 MHz, CDCl₃) δ 8.1-8.0(d, J=8 Hz, 1H), 7.8(d, J=7.6 Hz, 1H),7.8-7.7(d, J=8 Hz, 1H), 7.5-7.4(m, 2H), 7.4-7.3(m, 2H), 4.1-4.0(m, 4H),3.1(d,J=17.6 Hz, 1H), 2.6(d, J=17.6 Hz, 1H), 1.8-1.7(m, 2H), 1.2(t, 3H),0.7(t, 3H)

EXAMPLE 26 t-Butyl(3S)-3-({[5-ethyl-3-(1-naphthylmethyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoate(Im)

The compounds of Preparations 18 and 10 were reacted according to thesame procedure as Preparation 4 and Example 1 to give the titlecompound.

¹H-NMR(400 MHz, CDCl₃) δ 8.0(m, 1H), 7.8(m, 1H), 7.8(m, 1H), 7.7(m, 1H),7.5(m, 2H), 7.4-7.3(m, 2H), 6.7(m, 1H), 5.0-4.9(m, 2H), 4.9-4.8(m, 1H),4.2-4.0(m, 2H), 3.1(dd, J=24.8 Hz, 1H), 2.9-2.6(m, 3H), 1.9(m, 1H),1.7(m, 1H), 1.4(two s, 9H), 0.8(m, 3H)

EXAMPLE 27(3S)-3-({[5-ethyl-3-(1-naphthylmethyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Inn)

The compound of Example 26 was reacted according to the same procedureas Example 2 to give the title compound.

¹H-NMR(400 MHz, DMSO-d₆) δ 12.3(br, 1H), 8.4(br, 1H), 8.0(m, 1H), 7.9(m,1H), 7.8(m, 1H), 7.5-7.4(m, 5H), 5.0(br, 2H), 4.7(m, 1H), 4.0(s, 2H),3.3(m, 1H), 3.1(m, 1H), 2.8(m, 2H), 2.5(m, 1H), 1.8(m, 1H), 1.7(m, 1H),0.8(m, 3H)

MS [M+H]⁺ 561

EXAMPLE 28(3S)-5-(t-butoxy)-3-[({5-ethyl-3-[2-(1-naphthyl)ethyl]-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-2,5-dioxopentyl2,6-dichlorobenzoate (In)

1-Naphthylpropionaldehyde and the compound of Preparation 6 were reactedaccording to the same procedure as Preparations 1, 2, 3, and 4, andExample 1 to give the title compound.

¹H-NMR(400 MHz, CDCl₃) δ 8.00(d, 1H), 7.85(m, 1H), 7.76(m, 2H),7.57-7.28(m, 7H), 7.34-7.27(m, 3H), 5.20-5.05(m, 2H), 4.94 & 4.88(two m,1H), 3.39-3.27(m, 3H), 2.97-2.72(m, 5H), 2.07(m, 1H), 1.86(m, 1H), 1.46& 1.45(two s, 9H), 1.00 & 0.96(two t, 3H)

EXAMPLE 29(3S)-5-[(2,6-dichlorobenzoyl)oxy]-3-[({5-ethyl-3-[2-(1-naphthyl)ethyl]-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-4-oxopentanoicacid (Ioo)

The compound of Example 28 was reacted according to the same procedureas Example 2 to give the title compound.

¹H-NMR(400 MHz, DMSO-d₆) δ 8.45(bd, 1H), 8.06(d, 1H), 7.90(d, 1H),7.77(d, 1H), 7.60-7.37(m, 7H), 5.21-5.03(m, 2H), 4.73(m, 1H), 3.06(m,2H), 2.65(bd, 4H), 1.91 & 1.74(two m, 2H), 0.84(m, 3H)

Preparation 19

Ethyl 5-ethyl-3-[(1-naphthyloxy)methyl]-4,5-dihydro-5-isoxazolecarboxylate (VIl)

2-Ntroethanol pyranyl derivative (see Synthesis, 1993, 12, 1206-1208)and ethyl 2-ethylacrylate were reacted and the resulting product wasdeprotected to give ethyl5-ethyl-(3-hydroxymethyl)-4,5-dihydro-5-isoxazolecarboxylate (23%). Thiscompound was subjected to bromination (CBr₄, PPh₃, 94%) and then reactedwith 1-naphthol (NaH, DMF, 82%) to give the title compound.

¹H-NMR(500 MHz, CDCl₃) δ 8.20(m, 1H), 7.81(d, 1H), 7.55-7.42(m, 3H),7.36(m, 1H), 6.88(d, 1H), 5.01(dd, 2H), 4.22(m, 2H), 3.61(d, 1H),3.07(d, 1H), 1.97(m, 2H), 1.27(t, 3H), 0.93(t, 3H)

EXAMPLE 30(3S)-3-[({5-Ethyl-3-[(1-naphthyloxy)methyl]-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Ipp)

The compounds of Preparations 19 and 10 were reacted according to thesame procedure as Preparation 4 and Examples 1 and 2 to give the titlecompound.

¹H-NMR(400 MHz, DMSO-d₆) δ 8.51(br, 1H), 8.13(m, 1H), 7.87(m, 1H),7.54-7.38(m, 5H), 7.02(m, 1H), 5.18-4.97(m, 4H), 4.71(m, 1H), 3.58(twod, 1H), 3.21(two d, 1H), 2.68(m, 2H), 1.97(m, 1H), 1.81(m, 1H), 0.86(twot, 3H)

Mass: M+H 577

EXAMPLE 31(3S)-3-{[(3-{[(4-Chloro-1-naphthyl)oxy]methyl}-5-ethyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Iqq)

The bromo derivative obtained as an intermediate during the process ofPreparation 19 and 4-chloro-1-naphthol were reacted according to thesame procedure as Preparation 19 to give ethyl5-ethyl-3-[(4-chloro-1-naphthyloxy)methyl]-4,5-dihydro-5-isoxazodecarboxylate,which was then reacted according to the same procedure as Preparation 4and Examples 1 and 2 to give the title compound.

¹H-NMR(400 MHz, DMSO-d₆) δ 8.65(m, 1H), 8.24-8.09(m, 2H), 7.78-7.35(m,4H), 7.06(m, 1H), 5.17-5.07(m, 4H), 4.82-4.69(m, 1H), 3.45(two d, 1H),3.23(two d, 1H), 2.83-2.52(m, 2H), 1.96-1.79(m, 2H), 0.86(two t, 3H)

Mass: M+H 611

Preparation 20

tert-Butyl (3S)-3-amino-4-hydroxybutanoate

To N-benzyloxycarbonyl-β-t-butylaspartic acid (3.0 g, 9.28 mmol) and NMM(1.12 ml, 1.1 Eq) was added dry tetrahydrofuran (20 ml) under nitrogenatmosphere. The reaction solution was kept at 0° C. Isobutylchloroformate (1.26 ml, 1.05 Eq) was added, and the mixture was stirredfor about 40 minutes. Thus obtained slurry was filtered under nitrogen,and the resulting solution was added to NaBH₄ (702 mg, 2.0 Eq) solution[methanol (10 ml)-dry tetrahydrofuran (20 ml)] at −78° C. The resultingmixture was slowly warmed to room temperature (two hours). The reactionwas stopped by acetic acid (2.3 ml), and the reaction solution wasdistilled under reduced pressure to remove methanol. The residue wasextracted with ethyl acetate (50 ml×2), washed with water and aqueoussodium chloride solution, dried (anhydrous Na₂SO₄), concentrated underreduced pressure, and separated-purified by column chromatography (ethylacetate-hexane, 1:5) to give an alcohol compound (2.52 g, Yield 88%).

The benzyloxycarbonyl group of the compound obtained above was removed(Pd/C) under hydrogen baboon for one hour to give the title compound(Yield 100%).

¹H-NMR(400 MHz, DMSO-d₆) δ 4.60(bs, 1H), 3.21(m, 2H), 2.96(m, 1H),2.41(dd, 1H), 2.03(dd, 1H), 1.40(s, 9H)

Preparation 21

tert-Butyl(3S)-4-hydroxy-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)butanoate(XVI)

The compound of Preparation 14 was reacted according to the sameprocedure as Preparation 4 to give an active carboxylic acid derivative(VIIf). A mixture of said active carboxylic acid derivative (VIIf, 224mg, 0.79 mmol), the amino alcohol derivative prepared in Preparation 20(166 mg, 1.2 Eq) and HATU (390 mg, 1.3 Eq) was cooled to 0° C.,triethylamine (0.33 mg, 3.0 Eq) was added in a solvent of DMF(5 ml), andthe resulting mixture was reacted for two hours. The solvent wasdistilled under reduced pressure, and the residue was extracted withethyl acetate (50 ml×2), washed with water, aqueous sodium bicarbonatesolution, and aqueous sodium chloride solution, dried (anhydrousNa₂SO₄), concentrated under reduced pressure, and purified by columnchromatography (60-70% EA/Hex) to give the title compound (330 mg, Yield95%).

¹H-NMR(400 MHz, CDCl₃) δ 9.16(d, 1H), 8.55(d, 1H), 7.85(d, 1H),7.73-7.70(m, 3H), 7.56(NH, d, 1H), 4.26(m, 1H), 4.12(d, 1H), 4.04(d,1H), 3.79(d, 1H), 3.75(t, 2H), 2.94(bs, 1H), 2.57(m, 2H), 2.41(m, 1H),1.10 & 1.07(two d, 6H)

EXAMPLE 32 6-(tert-Butyl)1-ethyl(2E,4S)-4-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-2-hexenedioate (Iq)

To the compound of Preparation 21 (107 mg, 0.24 mmol) and Dess-Martinreagent (153 mg, 1.5 Eq) was added dry dichloromethane (4 ml), and themixture was stirred for one hour at room temperature and concentratedunder reduced pressure. To the residue were added dry THF (3 ml) and(carboethoxymethylene)triphenylphosphorane) (108 mg, 1.3 Eq), and themixture was refluxed for two hours. The reaction mixture was extractedwith ethyl acetate (20 ml×2), washed with water, saturated aqueoussodium bicarbonate solution and aqueous sodium chloride solution, dried(anhydrous Na₂SO₄), concentrated under reduced pressure, andpreliminarily purified by prep-chromatography (40% ethyl acetate-hexane)to give the title compound (98 mg, 80%) with the diastereomer ratio of3:1.

¹H-NMR(500 MHz, CDCl₃) of major isomer δ 9.15(d, 1H), 8.54(d, 1H),7.86(d, 1H), 7.74-7.62(m, 4H), 6.93(dd, 1H), 6.00(dd, 1H), 4.98(m, 1H),4.19(qt, 2H), 4.02(d, 1H), 3.81(d, 1H), 2.58(m, 2H), 2.43(m, 1H),1.36(s, 9H), 1.27(t, 3H), 1.07 & 1.06(two d, 6H)

EXAMPLE 33(3S,4E)-6-ethoxy-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-6-oxo-4-hexenoicacid (Irr)

The compound of Example 32 was reacted according to the same procedureas Example 2 to give the title compound.

¹H-NMR(500 MHz, MeOD-d₃) of major isomer δ 9.04(d, 1H), 8.61(d, ₁H),8.52(d, 1H), 7.95(d, 1H), 7.81-7.68(m, 3H), 6.94(dd, 1H), 6.00(dd, 1H),5.01(m, 1H), 4.17(qt, 2H), 3.90(d, 1H), 3.82(d, 1H), 2.71(d, 2H),2.39(septet, 1H), 1.26(t, 3H), 1.07 & 1.06(two d, 6H)

MS: M+H 454

EXAMPLE 34 tert-Butyl(3S,4E)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-(methylsulfonyl)-4-pentenoate(Ir)

Anhydrous dichloromethane (4 ml) was added to the compound of Example 21(109 mg, 0.25 mmol) and Dess-Martin reagent (157 mg, 1.5 Eq), and themixture was stirred for one hour at room temperature and concentratedunder reduced pressure. The reaction was stopped by isopropyl alcohol(0.5 ml). The solid was removed by filtration under reduced pressureusing Celite. The filtrate was extracted with ethyl acetate (20 ml×2),washed with water, saturated aqueous sodium bicarbonate solution, andaqueous sodium chloride solution, dried (anhydrous Na₂SO₄), andconcentrated under reduced pressure to give an aldehyde compound (540mg, Yield 99%).

Diethyl methylsilfonomethanephosphonate (113 mg, 2.0 Eq, Synthesis,1969, 170) was dissolved in THF (3 ml), NaH (60% in mineral oil, 20 mg,2.0 Eq) and above obtained aldehyde compound were added, and the mixturewas refluxed for two hours. After extraction by ethyl acetate (20 ml×2),the extract was washed with water, saturated aqueous sodium bicarbonatesolution, and aqueous sodium chloride solution, dried (anhydrousNa₂SO₄), concentrated under reduced pressure, and purified byprep-chromatography (60% ethyl acetate/hexane) to give the titlecompound (52 mg, 41%) with the diastereomer ratio of 4:1.

¹H-NMR(500 MHz, CDCl₃) of major diastereomer δ 9.14(d, 1H), 8.55(d, 1H),7.86(d, 1H), 7.76-7.65(m, 4H), 6.92(dd, 1H), 6.57(dd, 1H), 5.03(m, 1H),4.02(d, 1H), 3.81(d, 1H), 2.94(s, 3H), 2.61(m, 2H), 2.41(septet, 1H),1.36(s, 9H), 1.10 & 1.08(two set of d, 6H)

EXAMPLE 35(3S,4E)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-(methylsulfonyl)-4-pentenoicacid (Iss)

The compound of Example 34 was reacted according to the same procedureas Example 2 to give the title compound.

¹H-NMR(500 MHz, MeOD-d₃) of major isomer δ 9.04(d, 1H), 8.52(d, 1H),7.95(d, 1H), 7.82-7.69(m, 3H), 6.90(dd, 1H), 6.71(dd, 1H), 5.06(m, 1H),3.86(m, 2H), 2.98(s, 3H), 2.76(d, 2H), 2.39(m, 1H), 1.08 & 1.06(two setof d, 6H)

MS: M+H 460

PREPERATION METHOD OF EXAMPLES 36-42

The compounds which were obtained by reacting (iso)quinoinealdehyde (or1- or 2-naphthaldehyde) and ethyl 2-ethyl acrylate (or ethyl 2-propylacrylate) according to the same procedure as Preparations 1, 2 and 3 (orPreparations 1 and 16) were purified, if necessary, by prep-HPLC usingchiral OD column to isolate active chiral isomers. The isolated activechiral isomers or inactive chiral isomers were hydrolyzed according tothe same procedure as Preparation 4, and the resulting compounds werereacted with t-butyl 3-amino-5-fluoro-4-hydroxypentanoate according tothe same procedure as Examples 1 and 2 to give the title compounds offollowing Examples 36-42.

EXAMPLE 365-Fluoro-3-({[(5S)-3-(1-isoquinolinyl)-5-propyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid) (Itt)

¹H-NMR(500 MHz, DMSO-d₆) δ 9.02(t, 1H), 8.61(m, 2H), 8.08(d, 1H),7.96(m, 1H), 7.85(t, 1H), 7.78(m, 1H), 5.05(bs, 2H), 4.73(m, 1H),3.97-3.92(dd, 1H), 3.70-3.65(two d, 1H), 2.90-2.70(two m, 2H), 2.03(m,1H), 1.91(m, 1H), 1.48(m, 1H), 1.32(m, 1H), 0.93(m, 3H)

Mass: M+H 416

EXAMPLE 37(3-({[(5S)-5-ethyl-3-(1-naphthyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid) (Iuu)

¹H-NMR(500 MHz, DMSO-d₆) δ 8.84(s, 1H), 8.59(bs, 1H), 8.02(m, 2H),7.75(s, 1H), 7.60(m, 3H), 5.01(bs, 2H), 4.73(m, 1H), 3.87-3.82(dd, 1H),3.70-3.65(two d, 1H), 2.82-2.73(two m, 2H), 2.05(m, 1H), 1.95(m, 1H),0.96(m, 3H)

Mass: M+H 401

EXAMPLE 38(3-({[(5S)-5-ethyl-3-(2-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid) (Ivv)

¹H-NMR(500 MHz, DMSO-d₆) δ 8.61(dd, 1H),8.41(m, 1H), 8.02(m, 3H),7.79(t, 1H), 7.65(t, 1H), 5.22-5.06(m, 1H), 4.72(m, 1H), 4.49-4.28(m,1H), 3.84(m, 1H), 3.54(m, 1H), 2.99-2.54(two m, 2H), 2.01(m, 1H),1.91,(m, 1H), 0.91(m, 3H)

Mass: M+H 402

EXAMPLE 39(3-({[(5R)-5-ethyl-3-(3-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid) (Iww)

¹H-NMR(400 MHz, CDCl₃) δ (9.25 & 9.16(two s, 1H), 8.08˜7.64(m, 5H),4.90-4.84(m, 1H), 4.79-4.54(m, 2H), 4.01-3.93(two d, 1H), 3.54(d, 1H),3.03-2.87(m, 2H), 2.27-2.19 & 2.10-2.01(two m, 2H), 1.10-1.06(m, 3H)

Mass: M+H 402

EXAMPLE 403-({[5-ethyl-3-(8-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid) (Ixx)

¹H-NMR(500 MHz, CDCl₃) δ 8.98 & 8.93(two m, 1H), 8.56-8.21(m, 1H),8.03-7.94(m, 2H), 7.73(bs, 1H), 7.60(m, 1H), 7.53-7.48(two m, 1H),4.94-4.64(m, 3H), 4.27-4.22(two d, 1H), 3.77(d, 1H), 3.07 & 2.92(two m,2H), 2.31-2.22(m, 1H), 2.12-2.05(m, 1H), 1.10(t, 3H)

Mass: M+H 402

EXAMPLE 413-({[5-ethyl-3-(3-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid) (Iyy)

¹H-NMR(400 MHz, CDCl₃) δ 9.32 & 9.25(two d, 1H), 8.16-8.03(m, 2H),7.83-7.73(m, 3H), 7.62-7.55(m, 1H), 4.95-4.76(m, 3H), 3.86(two d, 1H),3.45-3.39(two d, 1H), 3.17-3.01(two m, 1H), 2.90-2.82(m, 1H),2.27-2.20(m, 1H), 2.09-2.00(m, 1H), 1.10-1.05(m, 3H)

Mass: M+H 402

EXAMPLE 425-Fluoro-3-({[(5R)-5-isopropyl-3-(2-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid) (Izz)

¹H-NMR(400 MHz, CDCl₃) δ 8.24(m, 1H), 8.14(m, 1H), 8.04(d, 1H), 7.88(d,1H), 7.79(m, 1H), 7.65(m, 1H), 7.51(bs, 1H), 5.13-4.35(m, 3H),4.05-4.00(two d, 1H), 3.73(d, 1H), 3.05-2.82(m, 2H), 2.42(m, 1H),1.10(m, 6H)

Mass: M+H 416

PREPERATION METHOD OF EXAMPLES 43-48

The compounds obtained by reacting an a romatic aldehyde (Bioorg. Med.Chem. Lett., 1996, 6, p 2173, J. Org. Chem., 1978, 43, 1372) and ethyl2-ethyl acrylate (or ethyl 2-isopropyl acrylate) according to the sameprocedure as Preparations 1, 2 and 3 (or Preparations 1 and 16) werepurified, if necessary, by prep-HPLC using chiral OD column to isolateactive chiral isomers. The isolated active chiral isomers or inactivechiral isomers were hydrolyzed according to the same procedure asPreparation 4, and the restating compounds were reacted with t-butyl3-amino-5-fluoro-4-hydroxypentanoate according to the same procedure asExamples 1 and 2 to give the title compounds of following Examples43-48.

EXAMPLE 433-({[5-Ethyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa1)

¹H-NMR(500 MHz, DMSO-d₆) δ 8.53(bs, 1H), 7.40(m, 2H), 7.32(m, 1H),7.25(m, 1H), 5.02(bs, 2H), 4.72(m, 1H), 3.60-3.54(two d, 1H),3.46-3.43(two d, 1H), 3.40(m, 1H), 2.78-2.60(two m, 2H), 1.95(m, 1H),1.85(m, 1H), 1.14(s, 6H), 0.88(m, 3H)

Mass: M+H 393

EXAMPLE 443-[({3-[3-(tert-Butyl)phenyl]-5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa2)

¹H-NMR(400 MHz, DMSO-d₆) δ 8.56(dd, 1H), 7.67(s, 1H), 7.48(m, 1H),7.38(d, 2H), 5.24-5.12(q, 1H), 4.76-4.69(m, 1H), 4.59-4.26(m, 1H),3.68(m, 1H), 3.49-3.43(two d, 1H), 2.96-2.56(two m, 2H), 1.98(m, 1H),1.85(m, 1H), 1.29(s, 9H), 0.89(d, 3H)

Mass: M+H 407

EXAMPLE 453-[({3-[4-(tert-Butyl)phenyl]-5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa3)

¹H-NMR(500 MHz, DMSO-d₆) δ 8.52(bs, 1H), 7.58(m, 2H), 7.43(dd, 2H),5.10(bs, 2H), 4.71-4.65(m, 1H), 3.65-3.59(two d, 1H), 3.40-3.35(two d,1H), 2.86-2.60(two m, 2H), 1.94(m, 1H), 1.82(m, 1H), 1.24(s, 9H),0.86(m, 3H)

Mass: M+H 407

EXAMPLE 465-Fluoro-3-({[(5R)-5-isopropyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa4)

¹H-NMR(500 MHz, CDCl₃) δ 7.60-7.53(m, 1H), 7.41(m, 2H), 7.27-7.23(m,2H), 5.20-4.91(m, 1H), 4.71(m, 1H), 4.54-4.42(m, 1H), 3.69-3.64(two d,1H), 3.45-3.38(m, 2H), 3.12-2.77(m, 2H), 2.32(m, 1H), 1.22(d, 6H),1.05(d, 6H)

Mass: M+H 407

EXAMPLE 473-[({(5R)-3-[3-(tert-butyl)phenyl]-5-isopropyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa5)

¹H-NMR(500 MHz, CDCl₃) δ 7.69(s, 1H), 7.48(d, 1H), 7.41(d, 1H), 7.35(t,1H), 4.74-4.50(m, 3H), 3.69(d, 1H), 3.40(d, 1H), 3.06-2.77(m, 2H),2.32(m, 1H), 1.33(s, 9H), 1.04(m, 6H)

Mass: M+H 421

EXAMPLE 483-{[(3-[1,1′-biphenyl]-3-yl-5-isopropyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-5-fluoro-4-oxopentanoicacid (Iaa6)

3-Biphenylcarboxaldehyde (see Synthesis, 2003, 337) was used as astarting material to give the title comound.

¹H-NMR(500 MHz, DMSO-d₆) δ 8.42(bs, 1H), 7.87(d, 1H), 7.73-7.69(m, 4H),7.52(m, 1H), 7.45(m, 2H), 7.38(m, 1H), 5.03(bs, 2H), 4.63(m, 1H),3.73-3.57(m, 2H), 2.67-2.63(two m, 2H), 2.22(m, 1H), 0.89(m, 6H)

Mass: M+H 441

PREPARATION METHOD OF EXAMPLES 49-59

4-Substituted pyridine 2-carboxaldehydes were obtained by a method knownin U.S. Pat. No. 6,043,248, JOC 1982, 47, p 4315, and they were reactedaccording to the same procedure as Preparations 1, 2 and 3 (orPreparations 1 and 16). The resulting compounds were purified, ifnecessary, by prep-HPLC using chiral OD column to isolate active chiralisomers. The isolated active chiral isomers or inactive chiral isomerswere hydrolyzed according to the same procedure as Preparation 4, andthe resulting compounds were reacted with t-butyl3-amino-5-fluoro-4-hydroxypentanoate according to the same procedure asExamples 1 and 2 to give the title compounds of following Examples49-59.

EXAMPLE 493-({[5-Ethyl-3-(2-pyridinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa7)

¹H-NMR(500 MHz, DMSO-d₆) δ 12.41(bs, 1H), 8.61(s, 1H), 8.59-8.53(dd,1H), 7.87(m, 1H), 7.44(m, 1H), 5.19-5.04(two d, 1H), 4.70(m, 1H),4.45-4.27(m, 1H), 3.67(m, 1H), 3.42(m, 1H), 2.95-2.53(two m, 2H),1.96(m, 1H), 1.86(m, 1H), 0.85(m, 3H)

Mass: M+H 352

EXAMPLE 503-[({3-[4-(tert-Butyl)-2-pyridinyl]-5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa8)

¹H-NMR(400 MHz, CDC₃) δ 8.58(d, 1H), 7.90(s, 1H), 7.65(bs, 1H),7.45-7.39(m, 1H), 4.98-4.68(m, 3H), 3.92 & 3.88(two d, 1H), 3.52 &3.47(two d, 1H), 3.05-2.86(m, 2H), 2.23-2.18(m, 1H), 2.08-2.00(m, 1H),1.38(s, 9H), 1.05(t, 3H)

Mass: M+H 408

EXAMPLE 513-[({(5R)-3-[4-(tert-butyl)-2-pyridinyl]-5-isopropyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa9)

¹H-NMR(400 MHz, CDCl₃) δ 8.58(d, 1H), 7.88(d, 1H), 7.61(bs, 1H), 7.43(m,1H), 4.84(m, 1H), 4.60(bs, 2H), 3.89-3.82(two d, 1H), 3.60-3.55(two d,1H), 3.04-2.86(m, 2H), 2.38(m, 1H), 1.38(s, 9H), 1.07(m, 6H)

Mass: M+H 422

EXAMPLE 523-({[5-Ethyl-3-(4-isobutyl-2-pyridinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa10)

¹H-NMR(500 MHz, DMSO-d₆) δ 8.58-8.52(dd, 1H), 8.49(s, 1H), 7.68(s, 1H),7.27(s, 1H), 5.19-5.04(m, 1H), 4.69(m, 1H), 4.46-4.29(m, 1H), 3.68(m,1H), 3.41-3.37(dd, 1H), 2.93-2.46(m, 4H), 1.95-1.85(two m, 3H),0.86-0.82(m, 9H)

Mass: M+H 408

EXAMPLE 533-({[3-(4-Acetyl-2-pyridinyl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa11)

¹H-NMR(500 MHz, CDCl₃) δ 8.79(d, 1H), 8.35(s, 1H), 7.76(d, 1H), 7.46(bs,1H), 4.84-4.40(m, 3H), 3.90-3.85(two d, 1H), 3.51-3.46(two d, 1H),3.04-2.84(m, 2H), 2.65(s, 3H), HHHkdkdk2.16(m, 1H), 2.02(m, 1H), 1.03(m,3H)

Mass: M+H 394

EXAMPLE 543-({[3-(4-Cyclopropyl-2-pyridinyl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa12)

¹H-NMR(500 MHz, DMSO-d₆) δ 12.39(bs, 1H), 8.41(t, 1H), 7.56(d, 1H),7.12(m, 1H), 5.19-5.04(m, 1H), 4.72-4.66(m, 1H), 4.46-4.27(m, 1H),3.69-3.63(m, 1H), 3.39-3.35(two d, 1H), 2.80-2.74(m, 1H), 2.63-2.52(m,1H), 2.00-1.80(m, 3H), 1.06(m, 2H), 0.86-0.80(m, 5H)

Mass: M+H 392

EXAMPLE 55

3-({[3-(4-Cyclopentyl-2-pyridinyl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa13)

¹H-NMR(500 MHz, CDCl₃) δ 8.50(d, 1H), 7.67(m, 2H), 7.26(m, 1H),4.88-4.82(m, 1H), 4.65(bs, 2H), 3.88-3.81(two d, 1H), 3.45-3.41(two d,1H), 3.05(m, 1H), 2.96-2.80(m, 2H), 2.20-2.12(m, 3H), 2.02-1.96(m, 1H),1.83(m, 2H), 1.72(m, 2H), 1.60(m, 2H), 1.01(t, 3H)

Mass: M+H 420

EXAMPLE 563-({[(5R)-3-(4-cyclopentyl-2-pyridinyl)-5-isopropyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa14)

¹H-NMR(400 MHz, CDCl₃) δ 8.58(m, 1H), 7.74(d, 1H), 7.63(bs, 1H),7.38-7.32(dd, 1H), 4.88-4.81(m, 1H), 4.53(bs, 2H), 3.87-3.79(two d, 1H),3.56-3.53(two d, 1H), 3.12-3.04(m, 1H), 3.02-2.85(m, 2H), 2.38(m, 1H),2.16(m, 2H), 1.88(m, 2H), 1.78(m, 2H), 1.65(m, 2H), 1.07(m, 6H)

Mass: M+H 434

EXAMPLE 573-({[3-(4-Cyclohexyl-2-pyridinyl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa15)

¹H-NMR(400 MHz, CDCl₃) δ 8.61(m, 1H), 7.75 (d, 1H), 7.64(bs, 1H),7.39-7.33(dd, 1H), 4.86-4.53(two m, 3H), 3.87(t, 1H), 3.50-3.45(dd, 1H),3.05-2.85(two m, 2H), 2.63(m, 1H), 2.22(m, 1H), 2.03(m, 1H),1.93-1.80(two m, 5H), 1.44(m, 5H), 1.05(t, 3H)

Mass: M+H 434

EXAMPLE 583-({[5-Ethyl-3-(5,6,7,8-tetrahydro-1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa16)

H-NMR(500 MHz, DMSO-d₆) δ 8.60-8.53(dd, 1H), 8.30(d, 1H), 7.15(d, 1H),5.20-5.05(m, 1H), 4.74-4.66(m, 1H), 4.45-4.25(m, 1H), 3.75-3.68(m, 1H),3.47-3.41(m, 1H), 2.94-2.88(m, 2H), 2.80-2.72(m, 3H), 2.62-2.53(m, 1H),1.99-1.93(m, 1H), 1.88-1.80(m, 1H), 1.71-1.69(m, 4H), 0.88-0.84(m, 3H)

Mass: M+H 406

EXAMPLE 595-Fluoro-3-({[5-isopropyl-3-(4-phenyl-2-pyridinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa17)

¹H-NMR(400 MHz, CDCl₃) δ 8.68(d, 1H), 8.14(s, 1H), 7.68(m, 1H), 7.58(m,1H), 7.54-7.43(m, 5H), 4.86-4.60(m, 3H), 3.92-3.87(two d, 1H), 3.62(d,1H), 3.09-2.86(m, 2H), 2.39(m, 1H), 1.11-1.06(m, 6H)

Mass: M+H 442

PREPARATION METHOD OF EXAMPLES 60-63

(2S)-4-(tert-butoxy)-2-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxobutanoicacid [obtained by coupling reaction of Comound (VIIf) withAsp(O-t-Bu)-OMe and hydrolysis] was reacted according to the sameprocedure as Preparation 5 to give bromomethyl ketone derivative(tert-butyl(3S)-5-bromo-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoate).This bromomethyl ketone derivative was reacted with diphenylphosphinicacid in KF/DMF (see Preparation 5) to give diphenylphosphoryloxymethylketone derivative, which was then reacted according to the sameprocedure as Example 2 to give the title compound (Compound of Example60).

The compounds of following Examples 61-63 were obtained from1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-ol,3-benzyl-4-hydroxy-2(5H)-furanone, or isobutyric acid according to thesame procedure as above.

EXAMPLE 60(3S)-5-[(diphenylphosphoryl)oxy]-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoic acid (Iaa18)

¹H-NMR(500 MHz, DMSO-d₆) δ 8.92(m, 1H), 8.58(m, 1H), 8.05(m, 1H),7.94(m, 1H), 7.81(m, 1H), 7.75-7.72(m, 3H), 7.57(m, 3H), 7.49(m, 4H),7.35(m, 2H), 4.90-4.65(two m, 3H), 3.79-3.75(two d, 1H), 3.69-3.67(twod, 1H), 2.65(two m, 2H), 2.22(m, 1H), 0.84(m, 6H)

Mass: M+H 614

EXAMPLE 61(3S)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-{[1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]oxy}pentanoicacid (Iaa19)

¹H-NMR(500 MHz, DMSO-d₆) δ 8.95(m, 1H), 8.83(d, 1H), 8.57(m, 1H),8.03(d, 1H), 7.92(d, 1H), 7.80(m, 1H), 7.74(m, 1H), 7.65(d, 1H), 7.57(m,1H), 7.51-7.34(m, 3H), 6.27(s, 1H), 5.29-5.15(m, 2H), 4.76(m, 1H),3.92-3.88(m, 1H), 3.75-3.69m, 1H), 2.84-2.60(m, 2H), 2.33(m, 1H),0.96(m, 6H)

Mass: M+H 624

EXAMPLE 62(3S)-5-[(4-benzyl-5-oxo-2,5-dihydro-3-furanyl)oxy]-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoic acid (Iaa20)

¹H-NMR(500 MHz, DMSO-d₆) δ 8.97(t, 1H), 8.58(d, 1H), 8.04(d, 1H),7.92(d, 1H), 7.81(m, 1H), 7.73(m, 1H), 7.23-7.07(m, 6H), 7.51(m, 1H),4.79-4.65(m, 3H), 3.92-3.87(two d, 1H), 3.74-3.68(two d, 1H),2.82-2.58(m, 2H), 2.31(m, 1H), 0.95(m, 6H)

Mass: M+H 586

EXAMPLE 63(3S)-5-(isobutyryloxy)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoic acid (Iaa21)

¹H-NMR(500 MHz, DMSO-d₆) δ 12.39(bs, 1H), 8.96(t, 1H), 8.73(m, 1H),8.58(dd, 1H), 8.04(d, 1H), 7.92(d, 1H), 7.81(t, 1H), 7.73(m, 1H),4.78(m, 2H), 4.68(m, 1H), 3.87(t, 1H), 3.73-3.68(two d, 1H),2.79-2.60(two m, 2H), 2.56(m, 1H), 2.31(m, 1H), 1.05(d, 3H), 0.95(m, 9H)

Mass: M+H 484

EXAMPLE 64(3S)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-hexenoicacid (Iaa22)

Weinreb amide derivative of(2S)-4-(tert-butoxy)-2-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxobutanoicacid (condensed derivative with N,O-dimethylhydroxylamine) and vinylmagnesium bromide were reacted, and the resulting compound was reactedaccording to the same procedure as Example 2 to give the title compound.

¹H-NMR(400 MHz, DMSO-d₆) δ 12.27(bs, 1H), 8.95(t, 1H), 8.74-8.67(two d,1H), 8.58(m, 1H), 8.04(d, 1H), 7.92(d, 1H), 7.83-7.72(m, 2H), 6.47(m,1H), 6.22-6.11(two dd, 1H), 5.71-5.62(two dd, 1H), 4.77(m, 1H),3.90-3.79(m, 1H), 3.72-3.64(m, 1H), 2.82(m, 1H), 2.62-2.49(m, 1H),2.30(m, 1H), 0.94(m, 6H)

Mass: M+H 410

Preparation 22

t-Butyl (3S)-3-amino-4-hydroxy-5-(2-pyridinyloxy)pentanoate (VIIIf)

The bromomethyl ketone derivative X (3.2 g, 7.93 mmol), an intermediatein Preparation 5, was dissolved in benzene (30 ml), Ag₂CO₃ (2.62 g, 1.2Eq) and 2-hydroxypyridine (0.93 g, 1.2 Eq) were added, and the mixturewas stirred under reflux at 80° C. for 3 days. The reaction mixture wasfiltered through Celite, concentrated under reduced pressure, andpurified by column chromatography to give 2-pyridinyloxymethyl ketonederivative (XIf) (466 mg, Yield 14%).

2-Pyridinyloxymethyl ketone derivative (XIf) (227 mg, 0.548 mmol) thusobtained was dissolved in a solvent mixture of tetrahydrofuran/methanol(3:2 v/v) (10 ml), NaBH₄ (43 mg, 2.0 Eq) was added at 0° C., and themixture was stirred for 30 minutes at room temperature. The reaction wasstopped by saturated ammonium chloride solution, and the reactionsolution was concentrated under reduced pressure. The residue wasextracted with ethyl acetate, washed with water and aqueous sodiumchloride solution, dried (anhydrous Na₂SO₄), and concentrated underreduced pressure to give Compound (XIIf) (230 mg) where the ketone groupwas reduced to hydroxyl group.

The benzyloxycarbonyl group of the compound prepared above was removed(Pd/C) under hydrogen balloon for 40 minutes to give the title compound(155 mg, 99%).

PREPARATION METHOD OF EXAMPLES 65-66

Compound VIIf (or VIIu) and VIIf were reacted according to the sameprocedure as Examples 1 and 2 to give the title compounds of Examples65-66. Here, VIIurepresents5-ethyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolecarboxylicacid.

EXAMPLE 65(3S)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2-pyridinyloxy)pentanoicacid (Iaa23)

¹H-NMR(500 MHz, MeOH-d₄) δ 9.04(d, 1H), 8.50(d, 1H), 7.95(d, 1H),7.81(d, 1H), 7.77(t, 1H), 7.69(t, 1H), 7.59(m, 1H), 6.85-6.80(m, 2H),6.67(bs, 1H), 5.07(bs, 1H), 4.95(t, 1H), 4.84(m, 1H), 3.94-3.79(Abq,2H), 2.92-2.80(m, 2H), 2.39(m, 1H), 1.09(m, 6H)

Mass: M+H 491

EXAMPLE 66(3S)-3-({[5-ethyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2-pyridinyloxy)pentanoicacid (Iaa24)

¹H-NMR(500 MHz, DMSO-d₆) δ 8.71(bs, 1H), 8.02-7.94(two bs, 1H),7.71-7.64(m, 1H), 7.42-7.18(m, 4H), 6.96-6.90(two t, 1H), 6.85-6.80(twod, 1H), 4.99(bs, 2H), 4.86-4.78(m, 1H), 3.58(two d, 1H), 3.48(two d,1H), 3.36(m, 1H), 2.81-2.61(m, 2H), 2.05-1.80(two m, 2H), 1.14-1.03(m,6H), 0.93-0.88(m, 3H)

Mass: M+H 468.21

EXAMPLE 672-{[(3S)-4-carboxy-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-2-oxobutyl]oxy}-1-methylpyridiniumtrifluoromethanesulfonate (Iaa25)

The intermediate t-butyl((3S)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2-pyridinyloxy)pentanoate(49 mg, 90 μmol) obtained after the Dess-Martin oxidation reaction inExample 65 was dissolved in dichloromethane (2 ml), methyltrifluoromethanesulfonate (1.1 Eq, 10.1 μl) was added, and the mixturewas stirred for one day at room temperature. After concentration underreduced pressure, the residue was subjected to the same procedure asExample 2 to give the title compound (60 mg).

¹H-NMR(500 MHz, DMSO-d₆) δ 9.00-8.91(m, 2H), 8.76-8.57(m, 2H), 8.42(m,1H), 8.04(m, 1H), 7.93(m, 1H), 7.80(m, 1H), 7.75(m, 1H), 7.54(m, 1H),5.77-5.48(m, 2H), 4.85-4.81(m, 1H), 4.00(s, 3H), 3.94-3.69(m, 2H),2.87-2.60(m, 2H), 2.33(m, 1H), 0.98 & 0.94(two d, 6H)

Mass: M+505.20

EXAMPLE 682-{[(3S)-4-carboxy-3-({[5-ethyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-2-oxobutyl]oxy}-1-methylpyridiniumtrifluoromethane-sulfonate (Iaa26)

The intermediate t-butyl(3S)-3-({[5-ethyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2-pyridinyloxy)pentanoate(55mg, 0.105 mmol) obtained after the Dess-Martin oxidation reaction inExample 66 was dissolved in dichloromethane (2 ml), methyltri-fluoromethanesulfonate (1.1 Eq, 13 μl) was added, and the mixturewas stirred for one day at room temperature. After concentration underreduced pressure, the residue was subjected to the same procedure asExample 2 to give the title compound (59 mg, 94%).

¹H-NMR(500 MHz, DMSO-d₆) δ 8.80-8.73(two d, 1H), 8.65(m, 1H),8.45-8.38(m, 1H), 7.56-7.51(m, 2H), 7.43-7.31(m, 3H), 7.24-7.12(m, H),5.72-5.48(m, 2H), 4.91(m, 1H), 4.01 & 3.99(two s, 3H), 3.64(two d, 1H),3.48(two d, 1H), 3.42(m, 1H), 2.90-2.62(m, 2H), 2.05-1.80(two m, 2H),1.15-1.03(m, 6H), 0.93-0.88(m,3H).

Mass: M+482.17

PREPARATION METHOD OF EXAMPLES 69-70

Oxime derivative of 5-chloro-1-methyl-1H-indol-2-aldehyde or1,5-dimethyl-1H-indol-2-aldehyde was reacted with t-butyl3-amino-5-fluoro-4-hydroxypentanoate according to the same procedure asPreparations 1, 17, 4 and Examples 1, 2 to give the title compounds ofExamples 69-70.

EXAMPLE 693-({[3-(5-chloro-1-methyl-1H-indol-2-yl)-5-isopropyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa27)

¹H-NMR(500 MHz, CDCl₃) δ 7.59(s, 1H), 7.46-7.39(two d, 1H), 7.30-7.23(m,3H), 5.01(m, 1H), 4.75-4.46(m, 2H), 4.02(d, 1H), 3.95(s, 3H), 3.71(d,1H), 2.95(m, 1H), 2.77(m, 1H), 2.35(m, 1H), 1.04(m, 6H)

Mass: M+H 452

EXAMPLE 703-({[3-(1,5-dimethyl-1H-indol-2-yl)-5-isopropyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid) (Iaa28)

¹H-NMR(500 MHz, CDCl₃) δ 7.48(bs, 1H), 7.39(d, 1H), 7.23-7.14(m, 3H),4.76(m, 1H), 4.70-4.50(m, 2H), 4.07-4.02(two d, 1H), 3.99, 3.94 &3.93(three s, 3H), 3.75-3.48(m, 1H), 2.94-2.82(two m, 1H), 2.46-2.43(twos, 3H), 2.34(m, 1H), 1.05(m, 6H)

Mass: M+H 432

Preparation 23

Resolution of(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazole carboxylicacid

5-Isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolecarboxylic acidwas dissolved in acetone (190 ml) by heating at 60° C., and(S)-a-methybenzylamine (14.5 ml, 112 mmol) was added. The reactionmixture was coded to room temperature and stirred for 4 hours. Theresulting precipitate was filtered and washed three times by acetone (40ml).

Thus obtained (S)-a-methylbenzylamine salt of(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolecarboxylicacid was dissolved in the mixture (150 ml) of acetone and water (4:1) byheating at 90° C., cooled to room temperature, and stirred for 3 hours.The resulting precipitate was filtered and washed by acetone (40 ml)three times to give pure (S)-a-methylbenzylamine salt of(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolecarboxylcacid (12.8 g, 24.3%, 99.8% ee).

The optical purity of(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolecarboxylicacid was estimated by using ChiralCel OD column (4.6×250 mm, 5 mm). Themixture of n-hexane/isopropanol/trifluoroacetic acid (92/8/0.1) was usedas the mobile phase at the flow rate of 1 ml/min, the column temperaturewas 40° C., and the detection wavelength was 225 nm. The two opticalisomers were observed at the retention times of 7.8 min (R isomer) and21.1 min (S isomer), respectively.

EXAMPLE 71

Resolution of Compound (Iii-1)

1) Isolation of the SEED Compound

The two diastereomers of Example 16 were separated by using preparativeliquid chromatography silica column (diameter 40 mm, DYNAMAX-100 Å). Themixture of n-hexane/ethanol/trifluoroacetic acid (4000/32/2) was used asthe mobile phase at the flow rate of 50 ml/min, and the desireddiastereomer was collected with the ratio of 99:1 during the retentiontime of 62˜66 min at the detection wavelength of 227 nm (the twodiastereomers were broadly eluted with the continuously changed ratiountil 86 min). Thus obtained diastereomer of the 99:1 ratio wasdeprotected and used as the seed for the resolution.

The optical purity was estimated by using Atlantis C₁₈ column (4.6×250mm, 5 mm). The mixture of acetonitrile/water/trifluoroacetic acid wasused as the mobile phase at the flow rate of 1 ml/min according to thefollowing gradient:

t=0 min, 0:100:0.1

3 min, 20:80:0.1

50 min, 100:0:0.1

Under the detection wavelength of 227 nm, the two optical isomers wereobserved at the retention times of 36.6 min (3R, 5R isomer) and 37.1 min(3S, 5R isomer), respectively.

(3S, 5R isomer): tert-butyl(3S)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoate

¹H-NMR(500 MHz, CDCl₃) δ 9.13(d, 1H), 8.55(d, 1H), 7.85(d, 1H),7.78-7.65(m, 4H), 5.22-5.02(two m, 2H), 4.90(m, 1H), 4.02(d, 1H),3.80(d, 1H), 2.92-2.88(dd, 1H), 2.79-2.75(dd, 1H), 2.41(m, 1H), 1.34(s,9H), 1.09(m, 6H)

Mass: M+H 472

(3R, 5R isomer): tert-butyl(3R)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoate

¹H-NMR(400 MHz, CDCl₃) δ 9.14(d, 1H), 8.56(d, 1H), 7.87(d, 1H),7.77-7.64(m, 4H), 5.25-5.00(two q, 2H), 4.90(m, 1H), 4.03(d, 1H),3.82(d, 1H), 2.93(dd, 1H), 2.77(dd, 1H), 2.42(m, 1H), 1.35(s, 9H),1.11(m, 6H)

Mass: M+H 472

(3S)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoic acid (Iii-1)

¹H-NMR(400 MHz, CDCl₃) δ 9.07(d, 1H), 8.57(d, 1H), 7.89(d, 1H),7.78-7.69(m, 3H), 7.69(d, 1H), 4.88-4.79(m, 2H), 4.67(bs, 1H), 4.07(d,1H), 3.81(d, 1H), 3.08-2.93(m, 1H), 2.91-2.82(m, 1H), 2.43(m, 1H),1.12(m, 6H)

Mass: M+H 416

(3R)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoic acid (Iii-2)

¹H-NMR(500 MHz, CDCl₃) δ 9.07(d, 1H), 8.55(d, 1H), 7.87(d, 1H),7.75-7.68(m, 3H), 7.52(bs, 1H), 5.20-4.35(m, 3H), 4.02(d, 1H), 3.80(d,1H), 3.13-2.90(m, 2H), 2.39(m, 1H), 1.08(m, 6H)

Mass: M+H 416

2) Recrystallization

(3S)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo pentanoic acid (Iii-1)

Crude t-butyl5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoate(366 g) was dissolved in methylene chloride (1.9 kg) and coded to 0° C.Triethylsilane (235 g) and trifluoroacetic acid (1.88 kg) were added andstirred for one hour at 0° C. The reaction mixture was distilled underreduced pressure, redissolved in ethyl acetate (2.4 kg), and the pH wasadjusted to 5.2 by adding 2N aqueous sodium hydroxide solution (3.1 L).The organic layer was separated and the aqueous layer was extracted oncewith ethyl acetate (2.4 kg). Combined organic layer was distilled underreduced pressure to remove the solvent, the resulting residue wasdissolved in methyl t-butyl ether (530 g), and small amount ofcrystalline(3S)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazdyl]carbonyl}amino)-4-oxopentanoicacid was added. After stirring for 16 hours at room temperature,resulting precipitate was filtered and washed with the 9:1 mixture ofmethyl t-butyl ether and n-hexane (210 ml).

Thus obtained(3S)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazdyl]carbonyl}amino)-4-oxopentanoicacid was dissolved in ethyl acetate (240 ml) by heating at 50° C.,n-hexane (240 ml) was slowly added, and gradually cooled to roomtemperature. After stirring for 24 hours at room temperature, theresulting precipitate was filtered and washed with the 1:1 mixture ofethyl acetate and n-hexane (30 ml) to give the title compound (Iii-1,46.0 g, 3S/3R=94/6.0). The optical purity was estimated using Lunacolumn (4.6×250 mm, 5 mm, C₁₈). The mixture of acetonitrile/20 mM NH₄OAc(pH 6) (30/70) was used as the mobile phase at the flow rate of 1ml/min. Under the detection wavelength of 227 nm, the two opticalisomers were observed at the retention times of 8.0 min (3R isomer) and9.0 min (3S isomer), respectively.

Experiment 1

Determination of the Caspase Inhibitory Effect

Caspase-1 and caspase-8 known as cysteine proteases in the form ofα_(2β) ₂ were expressed, purified, and activated by modifying a methodknown in Thornberry, N. A. et al, Nature, 1992, 356, 768. Thornberry, N.A. Methods in Enzymology, 1994, 244, 615. Walker, N. P. C. et al. Cell,1994, 78, 343, and caspase-9 was also purified by a similar method, andthe inhibitory activity against them was tested. Briefly describing, p10and p20 subunits (Thornberry, N. A. et al, Nature, 1992, 356, 768) wereexpressed in E.coli and purified by nickel column and anionic exchangechromatography to give caspase-1, caspase-8 and caspase-9. Thefluorescent substrate AcYVAD-AFC for thus obtained caspase-1, AcDEVD-AFCfor caspase-8, and AcLEHD-AFC for caspase-9 were used for determiningspecific activity of the synthesized inhibitors. The enzyme reaction wascarried out at 25° C. with various concentrations of the inhibitors in abuffer solution containing 50 mM HEPES(pH 7.50), 10% (w/v) sucrose, 0.1%(w/v) CHAPS, 100 mM NaCl, 1 mM EDTA, and 10 mM DTT in the preserice of50 μM AcYVAD-AFC for 10 nM caspase-1, 50 μM AcDEVD-AFC for 2.1 nMcaspase-8, and 150 μM AcLEHD-AFC for 200 nM caspase-9. The inhibitoryconstants K_(i) and K_(obs) of the inhibitors were determined bymeasuring the reaction velocity with the time lapse using a fluorescentspectrometer and by obtaining the initial rate constant. K_(i) wascalculated from the Lineweaver Burk Plot, and K_(obs) from the followingEquation 1.K _(obs) =−ln(1−A _(r) /A _(oo))/t   Equation 1

in which

A_(r) means cleavate rate (%) at time t, and

A_(oo) means the maximum cleavage rate (%).

Spectra MAX GeminiXS Fluorescent Spectrometer of Molecular Device Co.was used at the excitation wavelength of 405 nm and the emissionwavelength of 505 nm.

The in vivo inhibitory activity of the inhibitors was determined bysubjecting Jurkat cell (ATCC TIB-152) to apoptosis using Fas antibody(Upstate Biotech 05-201) and by detecting the color change according tothe WST-1 method known in Francoeur A .M. and Assalian A. (1996)Biochemica 3, 19-25 to observe the amount of alive Jurkat cells when thecels were treated by the inhibitor. Spectra MAX 340 Spectrometer ofMolecular Device Co. was used at the absorbance wavelength of 440 nm.TABLE 1 Caspase-1 Caspase-8 Com. K_(obs)/[I] K_(obs)/[I] Caspase-9Jurkat cell No. (M⁻¹min⁻¹) (M⁻¹min⁻¹) K_(obs)/[I] (M⁻¹min⁻¹) IC₅₀ (μM)Icc 1.9E5 3.3E4 22.5 Idd 3.1E5 1.9E5 4.25 Iee 1.8E5 32.6 Iff 5.0E5 1.2Igg 2.7E6 1.5E6 3.2E5 0.17 Iii 1.1E6 1.3E7 2.0E5 0.1 Iii-u* 1.9E5 0.71Iii-1 2.3E7 Iii-2 4.7E5 Ijj 1.9E5 4.7 Ikk 1.2E5 1.1 Ill 1.3E5 39.2 Imm1.6E5 2.0 Inn 2.6E5 6.5 Ioo 2.0E4 50 Ipp 2.3E6 1.7E5 3.5 Iqq 3.3E6 1.4E537 Irr No Activity Iss No Activity Itt 4.3E5 0.16 Iuu 2.9E5 0.98 Ivv1.5E6 0.15 Iww 1.1E6 1.9 Ixx 4.0E4 26.5 Iyy 3.1E5 31.2 Izz 4.2E5 1.25Iaa1 1.8E5 0.33 Iaa2 3.2E5 0.56 Iaa3 1.4E5 2.65 Iaa4 5.0E5 0.67 Iaa53.0E5 0.3 Iaa6 2.0E5 2.45 Iaa7 4.4E4 2.27 Iaa8 4.5E5 0.70 Iaa9 2.3E51.24 Iaa10 1.5E5 1.02 Iaa11 6.4E4 31 Iaa12 1.9E5 0.70 Iaa13 3.1E5 0.35Iaa14 4.1E5 0.79 Iaa15 4.3E5 0.72 Iaa16 6.2E5 0.73 Iaa17 1.8E5 1.14Iaa18 5.2E5 1.5 Iaa19 1.6E4 No Activity Iaa20 3.7E4 No Activity Iaa21 NoActivity No Activity Iaa22 No Activity Iaa23 2.9E4 No Activity Iaa24 NoActivity Iaa25 1.7E5 No Activity Iaa26 3.1E4 No Activity Iaa27 8.2E5 1.3Iaa28 1.8E6 0.6*Compound Iii-u is the (5S) form of Comound Iii.

Experiment 2

Therapeutic Effect for LPS-Induced Acute Hepatitis in Mouse

Step 1) Preparation of Blood Sample

Female Balb/c mice (6 weeks, Charles River Laboratory, Osaka, Japan)were kept under the conditions of 22° C., 55% of relative humidity, andfight-darkness cycle of 12 hours. Food and water were supplied adlibitum. In pyrogen-free saline were dissolved LPS (lipopolysaccaride)and D-galactosamine in concentrations of 0.4 mg/ml and 280 mg/ml,respectively, and their 1:1 mixture was injected to mice in the amountof 5 ml/kg. Immediately after the injection of LPS and D-galactosamine,vehicle (a mixture of PEG400:ethanol:Tween80=15:7.5:2.5 was diluted byfive times with saline) wherein the test compound is dissolved or thevehicle alone was intraperitoneally injected into the mice. After 8hours from the drug injection, blood samples were obtained from theirhearts.

Step 2: Determination of the Activity of Plasma Aminotransferase

The plasma ALT activity was determined for the hood samples obtained inStep 1 using ALT assay kit (Asan Pharm. Co., Seoul, Korea) according tothe manufacturer's instruction. As a result, it appeared that theinjection of LPS and D-galactosamine steeply increases the ALT activityin plasma, and the test compounds inhibit the increased enzyme activityin a dose-dependent manner. Based on these results, ED₅₀ values of thetest compounds were calculated using Prism software of GraphPad Co. togive 0.01-10 mg/kg.

Experiment 3

Inhibitory Effect Against Hepatic Fibrosis in SD Rats

Step 1) Induction of Hepatic Fibrosis and Administration

Male Spargue-Dawley rats (6 weeks, Korea Biolink) were kept under theconditions of 22° C., 55% of relative humidity, and fight-darkness cycleof 12 hours. Food and water were supplied ad libitum. For the ligationof biliary duct, the rats were anesthetized by inhalation of 1%halothane, abdomen of the rats were cut open, the distal and proximalbiliary ducts were ligated, the part between the ligation sites werecut, 2 ml of saline was injected, and sutured.

Test compound (Iii) was orally administered in a dosage of 3 mg/kg or 10mg/kg twice a day. That is, the compound was dissolved in a cosolvent(PEG400:ethanol:Tween80=15:7.5:2.5), diluted with phosphate buffer (pH7.4) by five times, and administered. To the control group wasadministered the solution without the test compound. The administrationwas made for 1 week after 1 week from the ligation of biliary duct.

Step 2: Preparation of Tissue Section and Sirius Red Staining

After the administration, the rats were sacrificed. The livers werefixed by 10% neutral formalin and a paraffin section of 5 μm thicknesswas prepared and stained by 0.1% Sirius red (Drect red 80, Sigma). Thetissue section was investigated by an optical microscope to observe thecollagen fibers stained in red, and the results are represented in FIG.1.

Experiment 4

Inhibitory Effect Against Apoptosis During the Bile Stagnation in SDRats

The test compound (Iii) was administered to the SD rats whose biliaryducts were ligated according to the procedure as Step 1 of Experiment 3.After 2 weeks from the operation, the rats were sacrificed. The liverswere fixed by 10% neutral formalin and a paraffin section of 5 μmthickness was prepared. TUNEL staining was carried out using ApopTagPeroxidase In Sit-up Apoptosis Detection kit (Chemicon) according to themanufacturer's instruction. More than 10 sites of the TUNEL stainedliver tissue were photographed in 200 magnifications without anyoverlap, of which result was shown in FIG. 2. The number of celssuffering apoptosis in each test group was counted and represented inthe following Table 2. In the following table, the Sham operation meansthe case where the biliary duct was exposed but not ligated, and the BDLoperation means the case where only the vehicle was administered afterthe ligation of biliary duct. TABLE 2 Inhibitory effect of compound(Iii) against hepatic cell apoptosis during the bile stagnation (Unit:TUNEL positive cells/field) BDL + Sham BDL Compound (Iii) BDL + Compound(Iii) operation operation 3 mg/kg 10 mg/kg 0.76 ± 0.15 16.7 ± 2.75 4.12± 1.3 5.66 ± 1.34

Conclusion

The effective inhibition of apoptosis (75% inhibition) by Compound (Iii)was shown by TUNEL staining in a model established by the induction ofhepatic fibrosis through the bile stagnation. In addition, the SiriusRed staining showed a severe fibrosis in the control group, but almostno fibrosis in the Compound (Iii) treated group. This resultdemonstrates the inhibitory effect of Compound (Iii) against hepaticfibrosis in rats, and so it is expected that Compound (Iii) can exhibitthe pharmacological activity of suppressing liver cirrhosis byinhibiting the apoptosis of hepatic cell in clinical application. Theapplication may be expanded not orgy to the hepatic cirrhosis by bilestagnation, but also to various hepatic diseases where hepatic fibrosisdue to the apoptosis is included.

Experiment 5

Antiphlogistic Effect in Rats

Male SD rats (6 weeks, Orient) were kept under the conditions of 22° C.,55% of relative humidity, and light-darkness cycle of 12 hours. Food andwater were supplied ad libitum. Before the induction of edema, the footvolume of each rat was measured by plethysmometer. Before the inductionof edema, vehicle (a mixture of PEG400:ethanol:Tween80=15:7.5:2.5 wasdiluted by ten times with phosphate buffer) wherein the test compound isdissolved or the vehicle alone was orally administered into the rats.Carrageenan (gamma) was dissolved in the concentration of 1% in safine,and subcutaneously injected into the right rear foot of the rats in thedosage of 100 μl. After 3 hours, the right foot volume was measuredagain by plethysmometer. The effect of the test compound was shown byedema inhibition (%) (see Table 3). The edema inhibition (%) wascalculated according to the following Equation 2.Edema Inhibition (%)=100×(Average of the increased foot volume in thevehicle-administered group−Increased foot volume in the testcompound-administered group)/Average of the increased foot volume in thevehicle-administered group   Equation 2

TABLE 3 Edema inhibition effect of Compound (Iii) Edema inhibitioneffect (%) Dosage (mg/kg) Average SEM 3 2.3 7.7 30 20.7 9.7 100 22.7 2.6

Experiment 6

Therapeutic Effect for Rheumatic Arthritis in Rats

Female Lewis rats (7 weeks, Charles River, Japan) were kept under theconditions of 22° C., 55% of relative humidity, and light-darkness cycleof 12 hours. Food and water were supplied ad libitum. Type II collagen(bovine) was dissolved in 0.05M acetic acid in a concentration of 2mg/mg at 4° C., and the same amount of Complete Freund's adjuvant wasadded to emulsify the solution at 4° C. 0.1 ml of thus emulsifiedsolution was subcutaneously injected into the rattail of the Lewis rats.After 7 days, the emulsified solution was injected again in the samemanner. From the date on which the second emulsified solution wasinjected, vehicle (a mixture of PEG400:ethanol:Tween80=15:7.5:2.5 wasdiluted by ten times with phosphate buffer) wherein the test compound isdissolved or the vehicle done was orally administered into the ratsevery day. On Day 14 from the first injection of the collagen-containingemulsified solution, the right rear foot volume was measured again byplethysmometer (see Table 4). The edema inhibition (%) was determined bythe following Equation 3.   Equation 3Edema Inhibition (%)=100×(Average of the increased foot volume in thevehicle-administered group on Day 14−Increased foot volume in the testcompound-administered group on Day 14)/Average of the increased footvolume in the vehicle-administered group on Day 14   Equation 3

Further, p-value was calculated by Student t-test from the increasedfoot volume on Day 14 to confirm the significance. TABLE 4 Foot edemainhibition effect Edema inhibition Dosage of Foot volume (ml) effect (%)Compound (Iii) Day 0 Day 14 Day 14 (mg/kg) Average SEM Average SEMAverage SEM 0 1.115 0.036 1.975 0.257 — 10 1.134 0.032 1.394* 0.089 69.810.8*p-value = 0.032

In the foot edema test in rats, Compound (Iii) showed a dose-dependentedema inhibition effect of about 2, 21, and 23% at a dosage of 3, 30,and 100 mg/kg, respectively. In the model of rheumatic arthritis, thefoot edema was decreased significantly (about 70% decrease) in theCompound (Iii)-administered group on Day 14 when the negative controlgroup showed the maximum edema. Therefore, it is considered thatCompound (Iii) has an inhibitory effect against the progress ofinflammation in an inflammatory disease model, and so can treat theautoimmune inflammatory diseases.

Experiment 7

Effect Comparison with the Known Compound

Compound (Iii) according to the present invention and LB84068MP(Compound 68) known in PCT/KR00/01047 (WO 01/21600) were tested and theresults are shown in the following Table 5. TABLE 5 LB84068MP IiiK_(obs)/[I] against caspase-8 1.02E6 (M⁻¹min⁻¹) 1.30E7 (M⁻¹min⁻¹) IC₅₀(Jurkat cell) 1.6 μM 0.1 μM or less ED₅₀ (LPS + Gal) 0.99 mg/kg 0.01mg/kg Solubility (pH = 7.44 0.93 mg/ml or less 7.8 g/ml phosphate)

As can be seen from the above Table 5, Compound (Iii) according to thepresent invention showed an increased inhibitory activity by 10 times ormore against caspase-8 and Jurkat cell compared with LB84068MP, and byabout 100 times against LPS-induced acute hepatitis model (mouse). Also,regarding the physico-chemical properties, the compound showed improvedsolubility, and so can be easily used as an injection. That is, thecompound of the present invention shows highly more improved effectsthan the comparison compound of LB84068MP in both aspects of activityand physico-chemical properties.

1. A compound of the following formula (1):

in which I) R represents H, simple alkyl chain (—SAC), simple cycloalkylchain (—SCAC), aryl group (—Ar), or simple alkyl chain substituted byaryl (—SAC—Ar), II) R¹ represents —SAC, —SCAC, —Ar, —SAC—Ar, or a sidechain residue of all the natural amino acids; and the compound offormula (1) may exist in a specific diastereomeric form, or mixturesthereof when the carbon to which R′ is attached becomes a stereocenterdue to the R¹ group; or the compound of formula (1) may have aprotecting group in an ester form (—CO₂R³ wherein R³ is —SAC) or asulfonamide form (—CONHSO₂R⁴ wherein R⁴ is —SAC), or may exist in theform of pharmaceutically acceptable salt, when R¹is a side chain residueof an amino acid containing carboxyl moiety; or the compound of formula(1) may also exist in the form of pharmaceutically acceptable salt whenR¹ is a side chain residue of an amino acid containing a base moiety,III) R² represents —SAC, —SCAC, —Ar, —SAC—Ar, or a side chain residue ofthe natural amino acids; and the compound of formula (1) may exist in aspecific diastereomeric form, or mixtures thereof when the carbon towhich R² is attached becomes a stereocenter due to the R² group; thecompound of formula (1) may have a protecting group in an ester form(—CO₂R⁵ wherein R⁵ is —SAC) or a sulfonamide form (—CONHSO₂R⁶ wherein R⁶is —SAC), or may exist in the form of pharmaceutically acceptable salt,when R² is a side chain residue of an amino acid containing carboxylmoiety; or the compound of formula (1) may also exist in the form ofpharmaceutically acceptable salt when R² is a side chain residue of anamino acid containing a base moiety, or R² further represents H;—(CH₂)_(n) OR⁷ wherein R⁷ is —SAC, —SCAC, —Ar, or —SAC—Ar, and n=1 or 2;or —(CH₂)_(n) OC(═O)R⁸ wherein R is —SAC, —SCAC, —Ar, or —SAC—Ar, andn=1 or 2, IV) A represents —(CH₂)_(n)-(n=0-4), —O—(CH₂)_(n)-(n=0-4), or—NR⁹—(CH₂)_(n)-(n=0-4) wherein R⁹ is —SAC, —SCAC, —Ar, or —SAC—Ar, V)Brepresents H, —SAC, —SCAC, —Ar, or —SAC—Ar, or VI) R and R¹ may form acycle together with the carbon atom to which they are attached, where—R—R¹— is —(CH₂)_(n)—, —(CH₂)_(n)—O—(CH₂)_(m)—, or—(CH₂)_(n)—NR¹⁰O—(CH₂)_(m)— wherein n+m<9 and R¹⁰ is —SAC, —SCAC, —Ar,—SAC—Ar, —C(═O)—SAC, —C(═O)—SCAC, —C(═O)—Ar, or —C(═O)—SAC—Ar, VII) Xrepresents —C(═O)CH₂OR¹¹ wherein R¹¹ is —SAC, —SCAC, —Ar, or —SAC—Ar;—C(═O)CH₂OC(═O)R¹² wherein R¹² is —SAC, —SCAC, —Ar, or —SAC—Ar;—CH═CH—CO₂R¹³ wherein R¹³ is —SAC, —SCAC, —Ar, or —SAC—Ar; —CH═CH—SO₂R¹⁴ wherein R¹⁴ is —SAC, —SCAC, —Ar, or —SAC—Ar; —C(═O)CH═CH₂; or—COCH₂—W wherein W is —N₂, —F, —Cl, —Br, —I, —NR¹⁵R¹⁶ (R¹⁵ and R¹⁶ eachare —SAC, —SCAC, —Ar, or —SAC—Ar, or together may form 3- to 6-memberedsaturated or unsaturated cyclic group), —SR¹⁷ (R¹⁷ is —SAC, —SCAC, —Ar,or —SAC—Ar), or is the following formula:

wherein Y is H, —OH, —OR¹⁸ (R¹⁸═—SAC or —SCAC), —C(═O)R¹⁹ (R¹⁹═—H, —SAC,or —SCAC), —F, —Cl, —Br, —I, —C, —NC, —N₃, —CO₂H, CF₃, —CO₂R²⁰ (R²⁰═—SACor —SCAC), —C(═O)NHR²¹ (R²¹═—SAC or —SCAC), or —C(═O)NR²² R²³ (R²² andR²³ each are —SAC, —SCAC, —Ar, or —SAC—Ar), R²⁴ is H, —SAC, —SAC—Ar, or—Ar, salt, or stereoisomer thereof.
 2. The compound according to claim 1wherein R represents H.
 3. The compound according to claim 1 wherein R¹represents —CH₂COOH, —CH₂ COOR³(R³═SAC), or —CH₂CONHSO₂R⁴ (R⁴═SAC). 4.The compound according to claim 1 wherein R² represents H, —SAC, —Ar, or—(CH₂)_(n) OR⁷ (R⁷═—SAC, —SCAC, —Ar, or —SAC—Ar, and n=1 or 2).
 5. Thecompound according to claim 1 wherein X represents —C(═O)CH₂OAr,—C(═O)CH₂OC(═O)Ar, or —OCH₂—W wherein W is —N₂, —F, —Cl, —Br, —I,—NR¹⁵R¹⁶ (R¹⁵ and R¹⁶ each are —SAC, —SCAC, —Ar, or —SAC—Ar, or togethermay form 3- to 6-membered saturated or unsaturated cyclic group), or—SR¹⁷ (R¹⁷ is —SAC, —SCAC, —Ar, or —SAC—Ar).
 6. The compound accordingto claim 1 wherein I) R represents H, II) R represents —CH₂COOH,—CH₂COOR³ (R³═SAC), or —CH₂CONHSO₂R⁴ (R⁴═SAC), III) R² represents H,—SAC, —Ar, or —(CH₂)_(n)OR⁷ (R⁷═—SAC, —SCAC, —Ar, or —SAC—Ar, and n=1 or2), IV) A represents —(CH₂)_(n)-(n=0-4) or —O—(CH₂)_(n)-(n=0-4), V) Brepresents H, —SAC, —SCAC, —Ar, or —SAC—Ar, VI) X represents —COCH₂N₂,—COCH₂F, —COCH₂Cl, —COCH₂Br, —COCH₂I, —COCH₂OAr, —COCH₂OCOAr or—COCH₂SR¹⁷(R¹⁷ is —SAC,—SCAC, —Ar or —SAC—Ar).
 7. The compound accordingto claim 1 which is selected from the following group: (1)(3S)-5-[(2,6-dichlorobenzoyl)oxy]-3-({[5-methyl-3-phenyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa); (2)(3S)-3-({[5-methyl-3-phenyl-4,5-dihydro-5-isoxazdyl]carbonyl}amino)-4-oxo-5-phenoxypentanoicacid (Ibb); (3)(3S)-3-({[5-ethyl-3-phenyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Icc); (4)(3S)-3-({[5-ethyl-3-(1-naphthyl)-4,5-dihydro-5-isoxazdyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Idd); (5)(3S)-3-({[5-ethyl-3-(2-naphthyl)-4,5-dihydro-5-isoxazdyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Iee); (6)(3S)-3-({[5-ethyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazdyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Iff); (7)3-({[5-ethyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Igg); (8) ethyl3-({[5-ethyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoate(Ihh); (9)5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iii); (10)3-({[5-ethyl-3-(4-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Ijj); (11)3-({[3-(benzothiophen-2-yl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Ikk); (12)(3S)-3-({[3-(1,3-dimethyl-1H-indol-2-yl)-5-ethyl-4,5-dihydro-5-isoxazdyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Ill); (13)3-({[3-(1,3-dimethyl-1H-indol-2-yl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Imm); (14)(3S)-3-({[5-ethyl-3-(1-naphthylmethyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Inn); (15)(3S)-5-[(2,6-dichlorobenzoyl)oxy]-3-[({5-ethyl-3-[2-(1-naphthyl)ethyl]-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-4-oxopentanoicacid (Ioo); (16)(3S)-3-[(}5-ethyl-3-[(1-naphthyloxy)methyl]-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Ipp); (17)(3S)-3-{[(3-{[(4-chloro-1-naphthyl)oxy]methyl}-5-ethyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoicacid (Iqq); (18)(3S,4E)-6-ethoxy-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-6-oxo-4-hexenoicacid (Irr); (19)(3S,4E)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-(methylsulfonyl)-4-pentenoicacid (Iss); (20)5-fluoro-3-({[(5S)-3-(1-isoquinolinyl)-5-propyl-4,5-dihydro-5-isoxazdyl]carbonyl}amino)-4-oxopentanoicacid (Itt); (21)3-({[(5S)-5-ethyl-3-(1-naphthyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iuu); (22)3-({[(5S)-5-ethyl-3-(2-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Ivv); (23)3-({[(5R)-5-ethyl-3-(3-isoquinolinyl)-4,5-dihydro-5-isoxazdyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iww); (24)3-({[5-ethyl-3-(8-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Ixx); (25)3-({[5-ethyl-3-(3-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iyy); (26)5-fluoro-3-({[(5R)-5-isopropyl-3-(2-quinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Izz); (27)3-({[5-ethyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa1); (28)3-[({3-[3-(tert-butyl)phenyl]-5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa2); (29)3-[({3-[4-(tert-butyl)phenyl]-5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa3); (30)5-fluoro-3-({[(5R)-5-isopropyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa4); (31)3-[({(5R)-3-[3-(tert-butyl)phenyl]-5-isopropyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa5); (32)3-{[(3-[1,1′-biphenyl]-3-yl-5-isopropyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-5-fluoro-4-oxopentanoicacid (Iaa6); (33)3-({[5-ethyl-3-(2-pyridinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa7); (34)3-[({3-[4-(tert-butyl)-2-pyridinyl]-5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa8); (35)3-[({(5R)-3-[4-(tert-butyl)-2-pyridinyl]-5-isopropyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoicacid (Iaa9); (36) 3-({[5-ethyl-3-(4-isobutyl-2-pyridinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoic acid (Iaa10); (37)3-({[3-(4-acetyl-2-pyridinyl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa11); (38)3-({[3-(4-cyclopropyl-2-pyridinyl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa12); (39)3-({[3-(4-cyclopentyl-2-pyridinyl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa13); (40)3-({[(5R)-3-(4-cyclopentyl-2-pyridinyl)-5-isopropyl-4,5-dihydro-5-isoxazoyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa14); (41)3-({[3-(4-cyclohexyl-2-pyridinyl)-5-ethyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa15); (42)3-({[5-ethyl-3-(5,6,7,8-tetrahydro-1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa16); (43)5-fluoro-3-({[5-isopropyl-3-(4-phenyl-2-pyridinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa17); (44)(3S)-5-[(diphenylphosphoryl)oxy]-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa18); (45)(3S)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-{[1-phenyl-3-(trifluoromethyl)-1H-pyrazol5-yl]oxy}pentanoicacid (Iaa19); (46)(3S)-5-[(4-benzyl-5-oxo-2,5-dihydro-3-furanyl)oxy-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa20); (47)(3S)-5-(isobutyryloxy)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iaa21); (48)(3S)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-hexenoicacid (Iaa22); (49)(3S)-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2-pyridinyloxy)pentanoicacid (Iaa23); (50)(3S)-3-(}[5-ethyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxo-5-(2-pyridinyloxy)pentanoicacid (Iaa24); (51)2-{[(3S)-4-carboxy-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-2-oxobutyl]oxy}-1-methylpyridiniumtrifluoromethanesulfonate (Iaa25); (52)2-{[(3S)-4-carboxy-3-({[5-ethyl-3-(2-isopropylphenyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-2-oxobutyl]oxy}-1-methylpyridinium trifluoromethanesulfonate(Iaa26); (53) 3-({[3-(5-chloro-1-methyl-1H-indol-2-yl)-5-isopropyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa27); (54)3-({[3-(1,5-dimethyl-1H-indol-2-yl)-5-isopropyl-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluoro-4-oxopentanoicacid (Iaa28); and (55)(3S)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iii-1).
 8. The compound according to claim 1 which is5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iii).
 9. A compound of the following formula (1a):

in which A, B, R, R¹, R², and X are defined as described in claim
 1. 10.The compound according to claim 9 which is(3S)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid (Iii-1).
 11. A process for preparing the compound (Iii-1) asdefined in claim 10, which comprises the steps of dissolving a mixtureof(3S)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid and(3R)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid in methyl t-butyl ether, adding a small amount of crystalline(3S)-5-fluoro-3-({[(5R)-5-isopropyl-3-(1-isoquinolinyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4-oxopentanoicacid as a seed material to give a crystal, and re-crystallizing thiscrystal from ethyl acetate/n-hexane solvent system.
 12. A therapeuticcomposition for preventing inflammation and apoptosis comprising thecaspase inhibitor compound of formula (1), salt, or stereoisomer thereofas defined in claim 1 as an active ingredient together with thepharmaceutically acceptable carrier.
 13. The composition according toclaim 12 for the treatment of dementia, cerebral stroke, brainimpairment due to AIDS, diabetes, gastric ulcer, cerebral injure byhepatitis, hepatitis-induced hepatic diseases, acute hepatitis,fulminant hepatic failure, liver cirrhosis, sepsis, organtransplantation rejection, rheumatic arthritis, or cardiac cellapoptosis due to ischemic cardiac diseases.
 14. The compositionaccording to claim 12 for the treatment of acute hepatitis or livercirrhosis.
 15. The composition according to claim 12 for the treatmentof rheumatic arthritis.
 16. The composition according to claim 12 whichis formulated as an oral preparation, an injection, or a patch.
 17. Thecomposition according to claim 12 comprising the compound (Iii) asdefined in claim 8 as an active ingredient.
 18. The compositionaccording to claim 12 comprising the compound (Iii-1) as defined inclaim 10 as an active ingredient.
 19. A process for preparing thetherapeutic composition for preventing inflammation and apoptosis asdefined in claim 12, comprising admixing the caspase inhibitor compoundof formula (1), salt, or stereoisomer thereof as defined in claim 1 withpharmaceutically acceptable carrier.
 20. A method for preventinginflammation and apoptosis, comprising administering an effective amountof the caspase inhibitor compound of formula (1), salt, or stereoisomerthereof as defined in claim 1 to a patient suffering from inflammationand apoptosis.
 21. A use of the caspase inhibitor compound of formula(1), salt, or stereoisomer thereof as defined in claim 1 for preventinginflammation and apoptosis.